AbstractIn this technical note, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense

a variety of cell lines to ensure uniformity and viability for cell-based applications. The addition of Histodenz™

maintained the cells in suspension in the source plate to provide a stable and homogeneous cell solution through

the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing and provides a robust,

automated solution for situations in which biological material is limited or non-contact cell dispensing is required.

High-Precision Cell Dispensing with the Labcyte Echo® Liquid Handler

TECHNICAL NOTE

John Lesnick, Aurore Lejeune-Dodge, Noël Ruppert, and Carl Jarman

Labcyte Inc. | Sunnyvale, CA

TECHNICAL NOTE | High-Precision Cell Dispensing with the Labcyte Echo® Liquid Handler D107

2

touchless — no tips, nozzles, or physical materials contact the sample as it moves from the source microplate to a destination plate, array, or other substrate. Echo Liquid Handlers use no tips, allowing users to save money and eliminate waste, carry-over effects, and cross-contamination. The Echo 555 Liquid Handler can transfer in 2.5 nL increments to allow miniaturization with unmatched accuracy and precision.

TECHNICAL NOTE High-Precision Cell Dispensing with the Labcyte Echo® Liquid Handler

Cell-based assays have become indispensable for both industry and academic research efforts. These assays are used in various applications across the drug discovery and development trajectory: from target identification and validation to compound screening and monitoring cellular events to determine efficacy and biosafety. Basic research relies on cell-based assays to elucidate mechanisms of cell proliferation, differentiation, and function. These assays are an important part of research and development, and successfully dispensing viable cells is a key requirement for using them.

With most approaches, delivering consistent numbers of cells across an assay has been a challenge. Cells can be maintained in suspension or can require a surface to adhere to thrive. Even when starting with a seemingly homogeneous stock, the need to normalize cell density across wells often results in higher quantities of cells being used, which requires more reagents to optimize the assay. The use of frozen stock cells for assays can be used to minimize day-to-day variations of assay results due to change in cell passage or percent confluence at harvest.

The Labcyte Echo Liquid Handlers revolutionize liquid transfer by using acoustic energy to transfer samples and reagents. Echo systems have an acoustic transducer that emits low-energy sound waves that propel droplets from a source plate to an inverted destination plate above. Droplets are retained in the destination plate by electrostatics and surface tension. Transfer with Echo liquid handlers is completely

INTRODUCTION

FIGURE 1: The Echo Liquid Handler and a depiction of the transfer process from source well to destination well as a result of sound waves being generated by the transducer.

The cell-dispensing process must be quick, accurate, and gentle enough to ensure cell viability.

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The liver cancer cell line Hep-G2 is an adherent cell line with epithelial morphology that was resuspended from cryopreserved stocks. The THP-1 cell line is an acute monocytic leukemia cell derived from peripheral blood and grows in suspension. The Echo Liquid Handler provides a robust method for non-contact cell dispensing without stress on the cells, unlike flow-through solenoid or peristaltic technologies. With a dead volume down to 15µL and the ability to multiplex dispensing, the Echo is an automated solution that works even when biological material is limited. It’s an ideal fit for workflows involving precious cells such as CTCs, clonal profiling at the single-cell level, and screening of patient samples for a higher quality of data. We present here a series of experiments illustrating the consistency of cell delivery using Echo liquid handlers across a variety of cell types, densities, and conditions.

TEC-D107-1.0 3

LABCYTE INC.

PC-3 Cells Dispensed from Active CultureCASE STUDY 1

• Echo Qualified Reservoir (Labcyte # ER-0050)

• PC-3 Cell Line (ATCC # CRL-1435)

• Growth Media (RPMI 1640 supplemented with 10% FBS and 100 U/ml Pen/Strep)

• Histodenz™ (Sigma # D2158)

• CellTiter-Glo Viability Assay (Promega #G7570)

• 1536 Well TC-Treated Plate (Corning #3726)

MATERIALS

METHODS

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from flasks with Versene at ~80% confluence, centrifuged into a pellet, and resuspended in growth media at 2x final concentration. Histodenz was dissolved in growth media at 2x final percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL of cell solution to a 1536 well plate followed by 1 µL of CTG. After 15 minutes of incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

ASSAY DEVELOPMENT

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology and will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0% to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2-fold dilutions from this stock solution.

Histodenz 0% 2.50% 5% 7.50% 10% 12% 14% 16% 18% 20%Mean 12520 15709 19857 17998 21545 26031 28727 30173 30068 29276Std. Deviation 8816 9999 6917 7934 4940 2251 1233 1757 1211 1196Std. Error 1971 2236 1547 1774 1105 503 276 393 271 267CV 70.41% 63.65% 34.83% 44.08% 22.93% 8.65% 4.29% 5.82% 4.03% 4.08%

Mean 8911 8837 11444 12626 11147 14830 15579 17057 17312 17801Std. Deviation 4974 6041 4075 3057 3142 1632 928 683 699 994Std. Error 1112 1351 911 684 703 365 208 153 156 222CV 55.82% 68.36% 35.60% 24.21% 28.19% 11.01% 5.96% 4.00% 4.03% 5.58%

Mean 4243 3889 4331 5224 6443 8081 8871 9609 9653 10450Std. Deviation 3078 3231 2420 2943 1185 538 764 709 558 936Std. Error 688 722 541 658 265 120 171 159 125 209CV 72.55% 83.07% 55.88% 56.33% 18.39% 6.66% 8.61% 7.38% 5.78% 8.95%

Mean 1266 1603 1944 2734 3499 4173 4424 4502 4685Std. Deviation 1127 1336 1438 953 406 398 268 268 300Std. Error 252 299 322 213 91 89 60 60 67CV 89.03% 83.33% 74.00% 34.87% 11.61% 9.53% 6.06% 5.96% 6.41%

1000

Cel

ls

/ Wel

l50

0 C

ells

/ W

ell

250

Cel

ls

/ Wel

l12

5 C

ells

/ W

ell

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

Establishing a Method for High Precision Cell Dispensing with the Echo® 500 Series of Liquid HandlersNoël Ruppert, John Lesnick, Carl Jarman, Aurore Lejeune-Dodge, Linda OrrenLabcyte Inc., Sunnyvale, CA, USA | www.labcyte.com

© 2016 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

RESULTS

SUMMARY

• The Echo liquid handler provides high-quality cell seeding across a range of cell densities and volumes of transfer.

• Using the Echo Reservoir minimizes dead volume.

• The miniaturized method in 1536-well plates and 2µL reaction volume reduces input requirements and reduces detection cost.

• The method described can be applied universally to other cell lines of interest.

• The quality of the Echo liquid handler transfer linearity provides potential for single cell applications.

We would like to thank Elina Huovari, Laura Turunen and Jani Saarela from the Institute of Molecular Medicine Finland (FIMM) for their support and developmental work which led to this publication.

TABLE 1: General statistical analysis of Histodenz / cell solution density matrix. 18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

FIGURE 5: Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

The Labcyte Echo 500 series liquid handlers revolutionize liquid transfer by using acoustic energy to eject fluids. Transfer with Echo Liquid Handlers is completely touchless—no tips or nozzles, and no material contacts the sample as it moves from source to destination. The elimination of tips when using the Echo Liquid Handler provides additional cost savings and eliminates waste, carry-over e� ects and cross-contamination. The Echo 525 Liquid Handler can transfer in 25 nL increments to allow miniaturization with accuracy and precision.

The Echo Liquid Handler

FIGURE 1: The Labcyte Echo 525 Liquid Handler.

MATERIALS AND METHODS

RESULTS

• Echo Qualifi ed Reservoir – Labcyte # ER-0050

• PC-3 Cell Line – ATCC # CRL-1435

• Growth Media – RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep

• Histodenz™ – Sigma # D2158

• CellTiter-Glo Viability Assay – Promega #G7570

• 1536 Well TC-Treated Plate – Corning #3726

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The cells will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0 to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2 fold dilutions from this stock solution.

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from fl asks with Versene at ~80% confl uence, centrifuged into a pellet and resuspended in growth media at 2x fi nal concentration. Histodenz was dissolved in growth media at 2x fi nal percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL cell solution to a 1536 well plate followed by 1 µL addition of CTG. After a 15 minute incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

FIGURE 4 (A–D): Assay Development Matrix to optimize cell dispense with the Echo Liquid Handler

FIGURE 2: The Echo Qualifi ed Reservoir is specifi cally designed for the Echo 525 Liquid Handler to improve the e ̈ ciency when transferring a single fl uid to many wells in a destination plate. The product’s 6-well design enables the transfer of over 15 milliliters from a single plate. The working volume for each chamber is 2.55 mL, enabling fl exible assay assembly.

The Echo Qualifi ed Reservoir

An Echo 525 Liquid Handler source plate designed to enable convenient assay assembly and e ̈ cient large reagent volume transfers. The Reservoir enables scientists performing genomic, proteomic and cell-based assays to dispense costly and precious reagents and samples with minimal dead volume.

INTRODUCTION

Cell-based assays have become indispensable for both industry and academic research e� orts. Cell-based assays are put to use in various applications across the drug discovery and development trajectory: from target identifi cation and validation, compound screening, as well as monitoring cellular events to e ̈ cacy and biosafety. Basic research relies on numerous cell-based assays to elucidate basic mechanisms of cell proliferation, di� erentiation and function. Since live cells are used, more biologically relevant data sets are acquired. This reveals the complex cell biology information associated with the compound and target in the cells.

Cell-based assays are clearly an important part of drug discovery and successful dispensing of viable cells is a key requirement. The cell dispense must be quick, accurate and gentle enough to ensure cell viability. Establishing cell stability and uniformity to enable a high quality start point for a cell-based assay is an example of just one of the common challenges that can be avoided by e� ective cell dispensing.

In this study, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense PC-3 cells, an adherent line, out of the Labcyte Echo Qualifi ed Reservoir into 1536-well assay plates. The results are measured with the CellTiter-Glo luminescent cell viability assay and read on the BMG LABTECH PHERAstar FS HTS microplate reader. The addition of Histodenz™ maintained the cells in suspension during the transfer step to provide cell stability and uniformity through the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing to ensure uniformity and viability. 1µL Cell s

1µL CTG

FIGURE 3: Workfl ow to optimize cell dispense with the Echo liquid handler.

RESULTS

TABLE 2: Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2,4 and 8.

FIGURE 6: Cell dispense linearity at 18% Histodenz. R2 = 0.999.

A 1E6 cell suspension in 18% Histodenz was transferred to the Echo Reservoir. The Echo Liquid Handler was used to dispense variable volumes of cell suspension to the destination plate (1000nL, 500nL, 250nL and 125nL). Wells were backfi lled to 1µL with 18% Histodenz solution followed by CTG addition and luminescence measured.

Cell Dispense Linearity

Lum 125 250 500 1000

125 7144 1.9 4.2 8.1

250 1.9 13873 2.2 4.2

500 4.2 2.2 29980 1.9

1000 8.1 4.2 1.9 58178

Cell Number per Well

Cell

Num

ber p

er W

ell

Cells per well

Lum

ines

cenc

e0

250

500

750

1000

0100002000030000400005000060000

R2=0.999

A

C

B

D

PC-3 Cell Dispense18% Histodenz

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 10 120

10000

20000

30000

40000

125 cells per well3 cells per drop

500 cells per well12 cells per drop

1000 cells per well25 cells per drop

250 cells per well6 cells per drop

FIGURE 2 u

Workflow to optimize cell dispensing with the Echo Liquid Handler.

4

FIGURE 4 u

Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

Histodenz 0% 2.50% 5% 7.50% 10% 12% 14% 16% 18% 20%Mean 12520 15709 19857 17998 21545 26031 28727 30173 30068 29276Std. Deviation 8816 9999 6917 7934 4940 2251 1233 1757 1211 1196Std. Error 1971 2236 1547 1774 1105 503 276 393 271 267CV 70.41% 63.65% 34.83% 44.08% 22.93% 8.65% 4.29% 5.82% 4.03% 4.08%

Mean 8911 8837 11444 12626 11147 14830 15579 17057 17312 17801Std. Deviation 4974 6041 4075 3057 3142 1632 928 683 699 994Std. Error 1112 1351 911 684 703 365 208 153 156 222CV 55.82% 68.36% 35.60% 24.21% 28.19% 11.01% 5.96% 4.00% 4.03% 5.58%

Mean 4243 3889 4331 5224 6443 8081 8871 9609 9653 10450Std. Deviation 3078 3231 2420 2943 1185 538 764 709 558 936Std. Error 688 722 541 658 265 120 171 159 125 209CV 72.55% 83.07% 55.88% 56.33% 18.39% 6.66% 8.61% 7.38% 5.78% 8.95%

Mean 1266 1603 1944 2734 3499 4173 4424 4502 4685Std. Deviation 1127 1336 1438 953 406 398 268 268 300Std. Error 252 299 322 213 91 89 60 60 67CV 89.03% 83.33% 74.00% 34.87% 11.61% 9.53% 6.06% 5.96% 6.41%

1000

Cel

ls

/ Wel

l50

0 C

ells

/ W

ell

250

Cel

ls

/ Wel

l12

5 C

ells

/ W

ell

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

Establishing a Method for High Precision Cell Dispensing with the Echo® 500 Series of Liquid HandlersNoël Ruppert, John Lesnick, Carl Jarman, Aurore Lejeune-Dodge, Linda OrrenLabcyte Inc., Sunnyvale, CA, USA | www.labcyte.com

© 2016 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

RESULTS

SUMMARY

• The Echo liquid handler provides high-quality cell seeding across a range of cell densities and volumes of transfer.

• Using the Echo Reservoir minimizes dead volume.

• The miniaturized method in 1536-well plates and 2µL reaction volume reduces input requirements and reduces detection cost.

• The method described can be applied universally to other cell lines of interest.

• The quality of the Echo liquid handler transfer linearity provides potential for single cell applications.

We would like to thank Elina Huovari, Laura Turunen and Jani Saarela from the Institute of Molecular Medicine Finland (FIMM) for their support and developmental work which led to this publication.

TABLE 1: General statistical analysis of Histodenz / cell solution density matrix. 18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

FIGURE 5: Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

The Labcyte Echo 500 series liquid handlers revolutionize liquid transfer by using acoustic energy to eject fluids. Transfer with Echo Liquid Handlers is completely touchless—no tips or nozzles, and no material contacts the sample as it moves from source to destination. The elimination of tips when using the Echo Liquid Handler provides additional cost savings and eliminates waste, carry-over e� ects and cross-contamination. The Echo 525 Liquid Handler can transfer in 25 nL increments to allow miniaturization with accuracy and precision.

The Echo Liquid Handler

FIGURE 1: The Labcyte Echo 525 Liquid Handler.

MATERIALS AND METHODS

RESULTS

• Echo Qualifi ed Reservoir – Labcyte # ER-0050

• PC-3 Cell Line – ATCC # CRL-1435

• Growth Media – RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep

• Histodenz™ – Sigma # D2158

• CellTiter-Glo Viability Assay – Promega #G7570

• 1536 Well TC-Treated Plate – Corning #3726

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The cells will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0 to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2 fold dilutions from this stock solution.

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from fl asks with Versene at ~80% confl uence, centrifuged into a pellet and resuspended in growth media at 2x fi nal concentration. Histodenz was dissolved in growth media at 2x fi nal percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL cell solution to a 1536 well plate followed by 1 µL addition of CTG. After a 15 minute incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

FIGURE 4 (A–D): Assay Development Matrix to optimize cell dispense with the Echo Liquid Handler

FIGURE 2: The Echo Qualifi ed Reservoir is specifi cally designed for the Echo 525 Liquid Handler to improve the e ̈ ciency when transferring a single fl uid to many wells in a destination plate. The product’s 6-well design enables the transfer of over 15 milliliters from a single plate. The working volume for each chamber is 2.55 mL, enabling fl exible assay assembly.

The Echo Qualifi ed Reservoir

An Echo 525 Liquid Handler source plate designed to enable convenient assay assembly and e ̈ cient large reagent volume transfers. The Reservoir enables scientists performing genomic, proteomic and cell-based assays to dispense costly and precious reagents and samples with minimal dead volume.

INTRODUCTION

Cell-based assays have become indispensable for both industry and academic research e� orts. Cell-based assays are put to use in various applications across the drug discovery and development trajectory: from target identifi cation and validation, compound screening, as well as monitoring cellular events to e ̈ cacy and biosafety. Basic research relies on numerous cell-based assays to elucidate basic mechanisms of cell proliferation, di� erentiation and function. Since live cells are used, more biologically relevant data sets are acquired. This reveals the complex cell biology information associated with the compound and target in the cells.

Cell-based assays are clearly an important part of drug discovery and successful dispensing of viable cells is a key requirement. The cell dispense must be quick, accurate and gentle enough to ensure cell viability. Establishing cell stability and uniformity to enable a high quality start point for a cell-based assay is an example of just one of the common challenges that can be avoided by e� ective cell dispensing.

In this study, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense PC-3 cells, an adherent line, out of the Labcyte Echo Qualifi ed Reservoir into 1536-well assay plates. The results are measured with the CellTiter-Glo luminescent cell viability assay and read on the BMG LABTECH PHERAstar FS HTS microplate reader. The addition of Histodenz™ maintained the cells in suspension during the transfer step to provide cell stability and uniformity through the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing to ensure uniformity and viability. 1µL Cell s

1µL CTG

FIGURE 3: Workfl ow to optimize cell dispense with the Echo liquid handler.

RESULTS

TABLE 2: Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2,4 and 8.

FIGURE 6: Cell dispense linearity at 18% Histodenz. R2 = 0.999.

A 1E6 cell suspension in 18% Histodenz was transferred to the Echo Reservoir. The Echo Liquid Handler was used to dispense variable volumes of cell suspension to the destination plate (1000nL, 500nL, 250nL and 125nL). Wells were backfi lled to 1µL with 18% Histodenz solution followed by CTG addition and luminescence measured.

Cell Dispense Linearity

Lum 125 250 500 1000

125 7144 1.9 4.2 8.1

250 1.9 13873 2.2 4.2

500 4.2 2.2 29980 1.9

1000 8.1 4.2 1.9 58178

Cell Number per Well

Cell

Num

ber p

er W

ell

Cells per well

Lum

ines

cenc

e

025

050

075

010

000

100002000030000400005000060000

R2=0.999

A

C

B

D

PC-3 Cell Dispense18% Histodenz

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 10 120

10000

20000

30000

40000

125 cells per well3 cells per drop

500 cells per well12 cells per drop

1000 cells per well25 cells per drop

250 cells per well6 cells per drop

PC-3 Cell Dispense18% Histodenz

TECHNICAL NOTE High-Precision Cell Dispensing with the Labcyte Echo® Liquid Handler

Histodenz 0% 2.50% 5% 7.50% 10% 12% 14% 16% 18% 20%Mean 12520 15709 19857 17998 21545 26031 28727 30173 30068 29276Std. Deviation 8816 9999 6917 7934 4940 2251 1233 1757 1211 1196Std. Error 1971 2236 1547 1774 1105 503 276 393 271 267CV 70.41% 63.65% 34.83% 44.08% 22.93% 8.65% 4.29% 5.82% 4.03% 4.08%

Mean 8911 8837 11444 12626 11147 14830 15579 17057 17312 17801Std. Deviation 4974 6041 4075 3057 3142 1632 928 683 699 994Std. Error 1112 1351 911 684 703 365 208 153 156 222CV 55.82% 68.36% 35.60% 24.21% 28.19% 11.01% 5.96% 4.00% 4.03% 5.58%

Mean 4243 3889 4331 5224 6443 8081 8871 9609 9653 10450Std. Deviation 3078 3231 2420 2943 1185 538 764 709 558 936Std. Error 688 722 541 658 265 120 171 159 125 209CV 72.55% 83.07% 55.88% 56.33% 18.39% 6.66% 8.61% 7.38% 5.78% 8.95%

Mean 1266 1603 1944 2734 3499 4173 4424 4502 4685Std. Deviation 1127 1336 1438 953 406 398 268 268 300Std. Error 252 299 322 213 91 89 60 60 67CV 89.03% 83.33% 74.00% 34.87% 11.61% 9.53% 6.06% 5.96% 6.41%

1000

Cel

ls

/ Wel

l50

0 C

ells

/ W

ell

250

Cel

ls

/ Wel

l12

5 C

ells

/ W

ell

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

Establishing a Method for High Precision Cell Dispensing with the Echo® 500 Series of Liquid HandlersNoël Ruppert, John Lesnick, Carl Jarman, Aurore Lejeune-Dodge, Linda OrrenLabcyte Inc., Sunnyvale, CA, USA | www.labcyte.com

© 2016 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

RESULTS

SUMMARY

• The Echo liquid handler provides high-quality cell seeding across a range of cell densities and volumes of transfer.

• Using the Echo Reservoir minimizes dead volume.

• The miniaturized method in 1536-well plates and 2µL reaction volume reduces input requirements and reduces detection cost.

• The method described can be applied universally to other cell lines of interest.

• The quality of the Echo liquid handler transfer linearity provides potential for single cell applications.

We would like to thank Elina Huovari, Laura Turunen and Jani Saarela from the Institute of Molecular Medicine Finland (FIMM) for their support and developmental work which led to this publication.

TABLE 1: General statistical analysis of Histodenz / cell solution density matrix. 18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

FIGURE 5: Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

The Labcyte Echo 500 series liquid handlers revolutionize liquid transfer by using acoustic energy to eject fluids. Transfer with Echo Liquid Handlers is completely touchless—no tips or nozzles, and no material contacts the sample as it moves from source to destination. The elimination of tips when using the Echo Liquid Handler provides additional cost savings and eliminates waste, carry-over e� ects and cross-contamination. The Echo 525 Liquid Handler can transfer in 25 nL increments to allow miniaturization with accuracy and precision.

The Echo Liquid Handler

FIGURE 1: The Labcyte Echo 525 Liquid Handler.

MATERIALS AND METHODS

RESULTS

• Echo Qualifi ed Reservoir – Labcyte # ER-0050

• PC-3 Cell Line – ATCC # CRL-1435

• Growth Media – RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep

• Histodenz™ – Sigma # D2158

• CellTiter-Glo Viability Assay – Promega #G7570

• 1536 Well TC-Treated Plate – Corning #3726

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The cells will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0 to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2 fold dilutions from this stock solution.

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from fl asks with Versene at ~80% confl uence, centrifuged into a pellet and resuspended in growth media at 2x fi nal concentration. Histodenz was dissolved in growth media at 2x fi nal percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL cell solution to a 1536 well plate followed by 1 µL addition of CTG. After a 15 minute incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

FIGURE 4 (A–D): Assay Development Matrix to optimize cell dispense with the Echo Liquid Handler

FIGURE 2: The Echo Qualifi ed Reservoir is specifi cally designed for the Echo 525 Liquid Handler to improve the e ̈ ciency when transferring a single fl uid to many wells in a destination plate. The product’s 6-well design enables the transfer of over 15 milliliters from a single plate. The working volume for each chamber is 2.55 mL, enabling fl exible assay assembly.

The Echo Qualifi ed Reservoir

An Echo 525 Liquid Handler source plate designed to enable convenient assay assembly and e ̈ cient large reagent volume transfers. The Reservoir enables scientists performing genomic, proteomic and cell-based assays to dispense costly and precious reagents and samples with minimal dead volume.

INTRODUCTION

Cell-based assays have become indispensable for both industry and academic research e� orts. Cell-based assays are put to use in various applications across the drug discovery and development trajectory: from target identifi cation and validation, compound screening, as well as monitoring cellular events to e ̈ cacy and biosafety. Basic research relies on numerous cell-based assays to elucidate basic mechanisms of cell proliferation, di� erentiation and function. Since live cells are used, more biologically relevant data sets are acquired. This reveals the complex cell biology information associated with the compound and target in the cells.

Cell-based assays are clearly an important part of drug discovery and successful dispensing of viable cells is a key requirement. The cell dispense must be quick, accurate and gentle enough to ensure cell viability. Establishing cell stability and uniformity to enable a high quality start point for a cell-based assay is an example of just one of the common challenges that can be avoided by e� ective cell dispensing.

In this study, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense PC-3 cells, an adherent line, out of the Labcyte Echo Qualifi ed Reservoir into 1536-well assay plates. The results are measured with the CellTiter-Glo luminescent cell viability assay and read on the BMG LABTECH PHERAstar FS HTS microplate reader. The addition of Histodenz™ maintained the cells in suspension during the transfer step to provide cell stability and uniformity through the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing to ensure uniformity and viability. 1µL Cell s

1µL CTG

FIGURE 3: Workfl ow to optimize cell dispense with the Echo liquid handler.

RESULTS

TABLE 2: Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2,4 and 8.

FIGURE 6: Cell dispense linearity at 18% Histodenz. R2 = 0.999.

A 1E6 cell suspension in 18% Histodenz was transferred to the Echo Reservoir. The Echo Liquid Handler was used to dispense variable volumes of cell suspension to the destination plate (1000nL, 500nL, 250nL and 125nL). Wells were backfi lled to 1µL with 18% Histodenz solution followed by CTG addition and luminescence measured.

Cell Dispense Linearity

Lum 125 250 500 1000

125 7144 1.9 4.2 8.1

250 1.9 13873 2.2 4.2

500 4.2 2.2 29980 1.9

1000 8.1 4.2 1.9 58178

Cell Number per Well

Cell

Num

ber p

er W

ell

Cells per well

Lum

ines

cenc

e

025

050

075

010

000

100002000030000400005000060000

R2=0.999

A

C

B

D

PC-3 Cell Dispense18% Histodenz

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 10 120

10000

20000

30000

40000

125 cells per well3 cells per drop

500 cells per well12 cells per drop

1000 cells per well25 cells per drop

250 cells per well6 cells per drop

FIGURE 3 u

Assay development matrix to optimize cell dispensing with the Echo Liquid Handler.

TABLE 1 u

General statistical analysis of Histodenz / cell solution density matrix.

18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

Histodenz 0% 2.50% 5% 7.50% 10% 12% 14% 16% 18% 20%Mean 12520 15709 19857 17998 21545 26031 28727 30173 30068 29276Std. Deviation 8816 9999 6917 7934 4940 2251 1233 1757 1211 1196Std. Error 1971 2236 1547 1774 1105 503 276 393 271 267CV 70.41% 63.65% 34.83% 44.08% 22.93% 8.65% 4.29% 5.82% 4.03% 4.08%

Mean 8911 8837 11444 12626 11147 14830 15579 17057 17312 17801Std. Deviation 4974 6041 4075 3057 3142 1632 928 683 699 994Std. Error 1112 1351 911 684 703 365 208 153 156 222CV 55.82% 68.36% 35.60% 24.21% 28.19% 11.01% 5.96% 4.00% 4.03% 5.58%

Mean 4243 3889 4331 5224 6443 8081 8871 9609 9653 10450Std. Deviation 3078 3231 2420 2943 1185 538 764 709 558 936Std. Error 688 722 541 658 265 120 171 159 125 209CV 72.55% 83.07% 55.88% 56.33% 18.39% 6.66% 8.61% 7.38% 5.78% 8.95%

Mean 1266 1603 1944 2734 3499 4173 4424 4502 4685Std. Deviation 1127 1336 1438 953 406 398 268 268 300Std. Error 252 299 322 213 91 89 60 60 67CV 89.03% 83.33% 74.00% 34.87% 11.61% 9.53% 6.06% 5.96% 6.41%

1000

Cel

ls

/ Wel

l50

0 C

ells

/ W

ell

250

Cel

ls

/ Wel

l12

5 C

ells

/ W

ell

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

Establishing a Method for High Precision Cell Dispensing with the Echo® 500 Series of Liquid HandlersNoël Ruppert, John Lesnick, Carl Jarman, Aurore Lejeune-Dodge, Linda OrrenLabcyte Inc., Sunnyvale, CA, USA | www.labcyte.com

© 2016 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

RESULTS

SUMMARY

• The Echo liquid handler provides high-quality cell seeding across a range of cell densities and volumes of transfer.

• Using the Echo Reservoir minimizes dead volume.

• The miniaturized method in 1536-well plates and 2µL reaction volume reduces input requirements and reduces detection cost.

• The method described can be applied universally to other cell lines of interest.

• The quality of the Echo liquid handler transfer linearity provides potential for single cell applications.

We would like to thank Elina Huovari, Laura Turunen and Jani Saarela from the Institute of Molecular Medicine Finland (FIMM) for their support and developmental work which led to this publication.

TABLE 1: General statistical analysis of Histodenz / cell solution density matrix. 18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

FIGURE 5: Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

The Labcyte Echo 500 series liquid handlers revolutionize liquid transfer by using acoustic energy to eject fluids. Transfer with Echo Liquid Handlers is completely touchless—no tips or nozzles, and no material contacts the sample as it moves from source to destination. The elimination of tips when using the Echo Liquid Handler provides additional cost savings and eliminates waste, carry-over e� ects and cross-contamination. The Echo 525 Liquid Handler can transfer in 25 nL increments to allow miniaturization with accuracy and precision.

The Echo Liquid Handler

FIGURE 1: The Labcyte Echo 525 Liquid Handler.

MATERIALS AND METHODS

RESULTS

• Echo Qualifi ed Reservoir – Labcyte # ER-0050

• PC-3 Cell Line – ATCC # CRL-1435

• Growth Media – RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep

• Histodenz™ – Sigma # D2158

• CellTiter-Glo Viability Assay – Promega #G7570

• 1536 Well TC-Treated Plate – Corning #3726

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The cells will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0 to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2 fold dilutions from this stock solution.

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from fl asks with Versene at ~80% confl uence, centrifuged into a pellet and resuspended in growth media at 2x fi nal concentration. Histodenz was dissolved in growth media at 2x fi nal percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL cell solution to a 1536 well plate followed by 1 µL addition of CTG. After a 15 minute incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

FIGURE 4 (A–D): Assay Development Matrix to optimize cell dispense with the Echo Liquid Handler

FIGURE 2: The Echo Qualifi ed Reservoir is specifi cally designed for the Echo 525 Liquid Handler to improve the e ̈ ciency when transferring a single fl uid to many wells in a destination plate. The product’s 6-well design enables the transfer of over 15 milliliters from a single plate. The working volume for each chamber is 2.55 mL, enabling fl exible assay assembly.

The Echo Qualifi ed Reservoir

An Echo 525 Liquid Handler source plate designed to enable convenient assay assembly and e ̈ cient large reagent volume transfers. The Reservoir enables scientists performing genomic, proteomic and cell-based assays to dispense costly and precious reagents and samples with minimal dead volume.

INTRODUCTION

Cell-based assays have become indispensable for both industry and academic research e� orts. Cell-based assays are put to use in various applications across the drug discovery and development trajectory: from target identifi cation and validation, compound screening, as well as monitoring cellular events to e ̈ cacy and biosafety. Basic research relies on numerous cell-based assays to elucidate basic mechanisms of cell proliferation, di� erentiation and function. Since live cells are used, more biologically relevant data sets are acquired. This reveals the complex cell biology information associated with the compound and target in the cells.

Cell-based assays are clearly an important part of drug discovery and successful dispensing of viable cells is a key requirement. The cell dispense must be quick, accurate and gentle enough to ensure cell viability. Establishing cell stability and uniformity to enable a high quality start point for a cell-based assay is an example of just one of the common challenges that can be avoided by e� ective cell dispensing.

In this study, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense PC-3 cells, an adherent line, out of the Labcyte Echo Qualifi ed Reservoir into 1536-well assay plates. The results are measured with the CellTiter-Glo luminescent cell viability assay and read on the BMG LABTECH PHERAstar FS HTS microplate reader. The addition of Histodenz™ maintained the cells in suspension during the transfer step to provide cell stability and uniformity through the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing to ensure uniformity and viability. 1µL Cell s

1µL CTG

FIGURE 3: Workfl ow to optimize cell dispense with the Echo liquid handler.

RESULTS

TABLE 2: Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2,4 and 8.

FIGURE 6: Cell dispense linearity at 18% Histodenz. R2 = 0.999.

A 1E6 cell suspension in 18% Histodenz was transferred to the Echo Reservoir. The Echo Liquid Handler was used to dispense variable volumes of cell suspension to the destination plate (1000nL, 500nL, 250nL and 125nL). Wells were backfi lled to 1µL with 18% Histodenz solution followed by CTG addition and luminescence measured.

Cell Dispense Linearity

Lum 125 250 500 1000

125 7144 1.9 4.2 8.1

250 1.9 13873 2.2 4.2

500 4.2 2.2 29980 1.9

1000 8.1 4.2 1.9 58178

Cell Number per Well

Cell

Num

ber p

er W

ell

Cells per well

Lum

ines

cenc

e

025

050

075

010

000

100002000030000400005000060000

R2=0.999

A

C

B

D

PC-3 Cell Dispense18% Histodenz

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 10 120

10000

20000

30000

40000

125 cells per well3 cells per drop

500 cells per well12 cells per drop

1000 cells per well25 cells per drop

250 cells per well6 cells per drop

PC-3 Cell Dispense1000 cells/well

PC-3 Cell Dispense500 cells/well

TEC-D107-1.0 5

LABCYTE INC.

TABLE 2 u

Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2, 4, and 8.

FIGURE 5 u

Cell dispense linearity at 18% Histodenz. R2 = 0.999.

Histodenz 0% 2.50% 5% 7.50% 10% 12% 14% 16% 18% 20%Mean 12520 15709 19857 17998 21545 26031 28727 30173 30068 29276Std. Deviation 8816 9999 6917 7934 4940 2251 1233 1757 1211 1196Std. Error 1971 2236 1547 1774 1105 503 276 393 271 267CV 70.41% 63.65% 34.83% 44.08% 22.93% 8.65% 4.29% 5.82% 4.03% 4.08%

Mean 8911 8837 11444 12626 11147 14830 15579 17057 17312 17801Std. Deviation 4974 6041 4075 3057 3142 1632 928 683 699 994Std. Error 1112 1351 911 684 703 365 208 153 156 222CV 55.82% 68.36% 35.60% 24.21% 28.19% 11.01% 5.96% 4.00% 4.03% 5.58%

Mean 4243 3889 4331 5224 6443 8081 8871 9609 9653 10450Std. Deviation 3078 3231 2420 2943 1185 538 764 709 558 936Std. Error 688 722 541 658 265 120 171 159 125 209CV 72.55% 83.07% 55.88% 56.33% 18.39% 6.66% 8.61% 7.38% 5.78% 8.95%

Mean 1266 1603 1944 2734 3499 4173 4424 4502 4685Std. Deviation 1127 1336 1438 953 406 398 268 268 300Std. Error 252 299 322 213 91 89 60 60 67CV 89.03% 83.33% 74.00% 34.87% 11.61% 9.53% 6.06% 5.96% 6.41%

1000

Cel

ls

/ Wel

l50

0 C

ells

/ W

ell

250

Cel

ls

/ Wel

l12

5 C

ells

/ W

ell

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

Establishing a Method for High Precision Cell Dispensing with the Echo® 500 Series of Liquid HandlersNoël Ruppert, John Lesnick, Carl Jarman, Aurore Lejeune-Dodge, Linda OrrenLabcyte Inc., Sunnyvale, CA, USA | www.labcyte.com

© 2016 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

RESULTS

SUMMARY

• The Echo liquid handler provides high-quality cell seeding across a range of cell densities and volumes of transfer.

• Using the Echo Reservoir minimizes dead volume.

• The miniaturized method in 1536-well plates and 2µL reaction volume reduces input requirements and reduces detection cost.

• The method described can be applied universally to other cell lines of interest.

• The quality of the Echo liquid handler transfer linearity provides potential for single cell applications.

We would like to thank Elina Huovari, Laura Turunen and Jani Saarela from the Institute of Molecular Medicine Finland (FIMM) for their support and developmental work which led to this publication.

TABLE 1: General statistical analysis of Histodenz / cell solution density matrix. 18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

FIGURE 5: Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

The Labcyte Echo 500 series liquid handlers revolutionize liquid transfer by using acoustic energy to eject fluids. Transfer with Echo Liquid Handlers is completely touchless—no tips or nozzles, and no material contacts the sample as it moves from source to destination. The elimination of tips when using the Echo Liquid Handler provides additional cost savings and eliminates waste, carry-over e� ects and cross-contamination. The Echo 525 Liquid Handler can transfer in 25 nL increments to allow miniaturization with accuracy and precision.

The Echo Liquid Handler

FIGURE 1: The Labcyte Echo 525 Liquid Handler.

MATERIALS AND METHODS

RESULTS

• Echo Qualifi ed Reservoir – Labcyte # ER-0050

• PC-3 Cell Line – ATCC # CRL-1435

• Growth Media – RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep

• Histodenz™ – Sigma # D2158

• CellTiter-Glo Viability Assay – Promega #G7570

• 1536 Well TC-Treated Plate – Corning #3726

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The cells will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0 to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2 fold dilutions from this stock solution.

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from fl asks with Versene at ~80% confl uence, centrifuged into a pellet and resuspended in growth media at 2x fi nal concentration. Histodenz was dissolved in growth media at 2x fi nal percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL cell solution to a 1536 well plate followed by 1 µL addition of CTG. After a 15 minute incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

FIGURE 4 (A–D): Assay Development Matrix to optimize cell dispense with the Echo Liquid Handler

FIGURE 2: The Echo Qualifi ed Reservoir is specifi cally designed for the Echo 525 Liquid Handler to improve the e ̈ ciency when transferring a single fl uid to many wells in a destination plate. The product’s 6-well design enables the transfer of over 15 milliliters from a single plate. The working volume for each chamber is 2.55 mL, enabling fl exible assay assembly.

The Echo Qualifi ed Reservoir

An Echo 525 Liquid Handler source plate designed to enable convenient assay assembly and e ̈ cient large reagent volume transfers. The Reservoir enables scientists performing genomic, proteomic and cell-based assays to dispense costly and precious reagents and samples with minimal dead volume.

INTRODUCTION

Cell-based assays have become indispensable for both industry and academic research e� orts. Cell-based assays are put to use in various applications across the drug discovery and development trajectory: from target identifi cation and validation, compound screening, as well as monitoring cellular events to e ̈ cacy and biosafety. Basic research relies on numerous cell-based assays to elucidate basic mechanisms of cell proliferation, di� erentiation and function. Since live cells are used, more biologically relevant data sets are acquired. This reveals the complex cell biology information associated with the compound and target in the cells.

Cell-based assays are clearly an important part of drug discovery and successful dispensing of viable cells is a key requirement. The cell dispense must be quick, accurate and gentle enough to ensure cell viability. Establishing cell stability and uniformity to enable a high quality start point for a cell-based assay is an example of just one of the common challenges that can be avoided by e� ective cell dispensing.

In this study, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense PC-3 cells, an adherent line, out of the Labcyte Echo Qualifi ed Reservoir into 1536-well assay plates. The results are measured with the CellTiter-Glo luminescent cell viability assay and read on the BMG LABTECH PHERAstar FS HTS microplate reader. The addition of Histodenz™ maintained the cells in suspension during the transfer step to provide cell stability and uniformity through the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing to ensure uniformity and viability. 1µL Cell s

1µL CTG

FIGURE 3: Workfl ow to optimize cell dispense with the Echo liquid handler.

RESULTS

TABLE 2: Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2,4 and 8.

FIGURE 6: Cell dispense linearity at 18% Histodenz. R2 = 0.999.

A 1E6 cell suspension in 18% Histodenz was transferred to the Echo Reservoir. The Echo Liquid Handler was used to dispense variable volumes of cell suspension to the destination plate (1000nL, 500nL, 250nL and 125nL). Wells were backfi lled to 1µL with 18% Histodenz solution followed by CTG addition and luminescence measured.

Cell Dispense Linearity

Lum 125 250 500 1000

125 7144 1.9 4.2 8.1

250 1.9 13873 2.2 4.2

500 4.2 2.2 29980 1.9

1000 8.1 4.2 1.9 58178

Cell Number per Well

Cell

Num

ber p

er W

ell

Cells per well

Lum

ines

cenc

e

025

050

075

010

000

100002000030000400005000060000

R2=0.999

A

C

B

D

PC-3 Cell Dispense18% Histodenz

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 10 120

10000

20000

30000

40000

125 cells per well3 cells per drop

500 cells per well12 cells per drop

1000 cells per well25 cells per drop

250 cells per well6 cells per drop

Histodenz 0% 2.50% 5% 7.50% 10% 12% 14% 16% 18% 20%Mean 12520 15709 19857 17998 21545 26031 28727 30173 30068 29276Std. Deviation 8816 9999 6917 7934 4940 2251 1233 1757 1211 1196Std. Error 1971 2236 1547 1774 1105 503 276 393 271 267CV 70.41% 63.65% 34.83% 44.08% 22.93% 8.65% 4.29% 5.82% 4.03% 4.08%

Mean 8911 8837 11444 12626 11147 14830 15579 17057 17312 17801Std. Deviation 4974 6041 4075 3057 3142 1632 928 683 699 994Std. Error 1112 1351 911 684 703 365 208 153 156 222CV 55.82% 68.36% 35.60% 24.21% 28.19% 11.01% 5.96% 4.00% 4.03% 5.58%

Mean 4243 3889 4331 5224 6443 8081 8871 9609 9653 10450Std. Deviation 3078 3231 2420 2943 1185 538 764 709 558 936Std. Error 688 722 541 658 265 120 171 159 125 209CV 72.55% 83.07% 55.88% 56.33% 18.39% 6.66% 8.61% 7.38% 5.78% 8.95%

Mean 1266 1603 1944 2734 3499 4173 4424 4502 4685Std. Deviation 1127 1336 1438 953 406 398 268 268 300Std. Error 252 299 322 213 91 89 60 60 67CV 89.03% 83.33% 74.00% 34.87% 11.61% 9.53% 6.06% 5.96% 6.41%

1000

Cel

ls

/ Wel

l50

0 C

ells

/ W

ell

250

Cel

ls

/ Wel

l12

5 C

ells

/ W

ell

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense1000 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

25000

30000

35000

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz

PC-3 Cell Dispense250 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

8000

10000

12000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense125 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

2000

4000

6000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

PC-3 Cell Dispense500 cells/well

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 100

5000

10000

15000

20000

2.5% Histodenz5% Histodenz7.5% Histodenz10% Histodenz

0% Histodenz 12% Histodenz14% Histodenz16% Histodenz18% Histodenz20% Histodenz

Establishing a Method for High Precision Cell Dispensing with the Echo® 500 Series of Liquid HandlersNoël Ruppert, John Lesnick, Carl Jarman, Aurore Lejeune-Dodge, Linda OrrenLabcyte Inc., Sunnyvale, CA, USA | www.labcyte.com

© 2016 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

RESULTS

SUMMARY

• The Echo liquid handler provides high-quality cell seeding across a range of cell densities and volumes of transfer.

• Using the Echo Reservoir minimizes dead volume.

• The miniaturized method in 1536-well plates and 2µL reaction volume reduces input requirements and reduces detection cost.

• The method described can be applied universally to other cell lines of interest.

• The quality of the Echo liquid handler transfer linearity provides potential for single cell applications.

We would like to thank Elina Huovari, Laura Turunen and Jani Saarela from the Institute of Molecular Medicine Finland (FIMM) for their support and developmental work which led to this publication.

TABLE 1: General statistical analysis of Histodenz / cell solution density matrix. 18% Histodenz provides high quality and precise cell seeding across all cell densities with 4-6% CV’s.

FIGURE 5: Precision over time. Cell titration with 18% Histodenz tested cell precision over time by plotting luminescence.

The Labcyte Echo 500 series liquid handlers revolutionize liquid transfer by using acoustic energy to eject fluids. Transfer with Echo Liquid Handlers is completely touchless—no tips or nozzles, and no material contacts the sample as it moves from source to destination. The elimination of tips when using the Echo Liquid Handler provides additional cost savings and eliminates waste, carry-over e� ects and cross-contamination. The Echo 525 Liquid Handler can transfer in 25 nL increments to allow miniaturization with accuracy and precision.

The Echo Liquid Handler

FIGURE 1: The Labcyte Echo 525 Liquid Handler.

MATERIALS AND METHODS

RESULTS

• Echo Qualifi ed Reservoir – Labcyte # ER-0050

• PC-3 Cell Line – ATCC # CRL-1435

• Growth Media – RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep

• Histodenz™ – Sigma # D2158

• CellTiter-Glo Viability Assay – Promega #G7570

• 1536 Well TC-Treated Plate – Corning #3726

The human prostate cancer cell line PC-3 is an adherent cell line with epithelial morphology. The cells will sediment over time in solution. A matrix of solutions containing the nonionic density gradient medium Histodenz was created to identify the most appropriate percentage (w/v) to create a solution that will maintain a cell suspension and reduce the sedimentation rate. A range of solutions from 0 to 20% was tested. Additionally, a range of cell densities was tested for each Histodenz % solution. The cell density titration included 1E6 cells per mL and four 2 fold dilutions from this stock solution.

PC-3 cells were maintained in growth media in log phase prior to plating. Cells were detached from fl asks with Versene at ~80% confl uence, centrifuged into a pellet and resuspended in growth media at 2x fi nal concentration. Histodenz was dissolved in growth media at 2x fi nal percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL cell solution to a 1536 well plate followed by 1 µL addition of CTG. After a 15 minute incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

FIGURE 4 (A–D): Assay Development Matrix to optimize cell dispense with the Echo Liquid Handler

FIGURE 2: The Echo Qualifi ed Reservoir is specifi cally designed for the Echo 525 Liquid Handler to improve the e ̈ ciency when transferring a single fl uid to many wells in a destination plate. The product’s 6-well design enables the transfer of over 15 milliliters from a single plate. The working volume for each chamber is 2.55 mL, enabling fl exible assay assembly.

The Echo Qualifi ed Reservoir

An Echo 525 Liquid Handler source plate designed to enable convenient assay assembly and e ̈ cient large reagent volume transfers. The Reservoir enables scientists performing genomic, proteomic and cell-based assays to dispense costly and precious reagents and samples with minimal dead volume.

INTRODUCTION

Cell-based assays have become indispensable for both industry and academic research e� orts. Cell-based assays are put to use in various applications across the drug discovery and development trajectory: from target identifi cation and validation, compound screening, as well as monitoring cellular events to e ̈ cacy and biosafety. Basic research relies on numerous cell-based assays to elucidate basic mechanisms of cell proliferation, di� erentiation and function. Since live cells are used, more biologically relevant data sets are acquired. This reveals the complex cell biology information associated with the compound and target in the cells.

Cell-based assays are clearly an important part of drug discovery and successful dispensing of viable cells is a key requirement. The cell dispense must be quick, accurate and gentle enough to ensure cell viability. Establishing cell stability and uniformity to enable a high quality start point for a cell-based assay is an example of just one of the common challenges that can be avoided by e� ective cell dispensing.

In this study, we demonstrate the utility of the Labcyte Echo Liquid Handler to accurately dispense PC-3 cells, an adherent line, out of the Labcyte Echo Qualifi ed Reservoir into 1536-well assay plates. The results are measured with the CellTiter-Glo luminescent cell viability assay and read on the BMG LABTECH PHERAstar FS HTS microplate reader. The addition of Histodenz™ maintained the cells in suspension during the transfer step to provide cell stability and uniformity through the dispense step. The Labcyte Echo Liquid Handler provides high-quality cell dispensing to ensure uniformity and viability. 1µL Cell s

1µL CTG

FIGURE 3: Workfl ow to optimize cell dispense with the Echo liquid handler.

RESULTS

TABLE 2: Cell dispense linearity at 18% Histodenz. Expected fold dilutions of 2,4 and 8.

FIGURE 6: Cell dispense linearity at 18% Histodenz. R2 = 0.999.

A 1E6 cell suspension in 18% Histodenz was transferred to the Echo Reservoir. The Echo Liquid Handler was used to dispense variable volumes of cell suspension to the destination plate (1000nL, 500nL, 250nL and 125nL). Wells were backfi lled to 1µL with 18% Histodenz solution followed by CTG addition and luminescence measured.

Cell Dispense Linearity

Lum 125 250 500 1000

125 7144 1.9 4.2 8.1

250 1.9 13873 2.2 4.2

500 4.2 2.2 29980 1.9

1000 8.1 4.2 1.9 58178

Cell Number per Well

Cell

Num

ber p

er W

ell

Cells per well

Lum

ines

cenc

e

025

050

075

010

000

100002000030000400005000060000

R2=0.999

A

C

B

D

PC-3 Cell Dispense18% Histodenz

Time (min)

Lum

ines

cenc

e

0 2 4 6 8 10 120

10000

20000

30000

40000

125 cells per well3 cells per drop

500 cells per well12 cells per drop

1000 cells per well25 cells per drop

250 cells per well6 cells per drop

RESULTS

The Echo Liquid Handler transfers PC-3 cells with precision and accuracy when maintained in Histodenz between 14% and 20%. The cv’s of cell transfer for PC-3 cells are lowest (4%-6%) when cells are resuspended in 18% Histodenz. The cell transfer was accurate at 1000, 500, 250 and 125 transferred cells per well at a constant volume and variable stock concentration. The Echo Liquid Handler transferred the PC-3 cells with precision at 1000, 500, 250 and 125 nL transfers of a constant stock solution and variable transfer volume. The linear correlation of these conditions yielded R2 of 0.999. This methodology can be applied to any cell line of interest to benefit from using the Echo Liquid Handler to transfer cells.

6

TECHNICAL NOTE High-Precision Cell Dispensing with the Labcyte Echo® Liquid Handler

Hep-G2 Cells Dispensed from Frozen Stock CASE STUDY 2

• Echo Qualified Reservoir (Labcyte # ER-0050)

• Hep-G2 Cell Line (ATCC HB-8065)

• Growth Media (RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep)

• Histodenz™ (Sigma # D2158)

• CellTiter-Glo Viability Assay (Promega #G7570)

• 1536 Well TC-Treated Plate (Corning #3726)

MATERIALS

METHODS

Hep-G2 cells were maintained in growth media in log phase prior to cryopreservation. Cells were thawed at 37oC and resuspended in media, centrifuged into a pellet, and resuspended in growth media at 2x final concentration. Histodenz was dissolved in growth media at 2x final percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL of cell solution to a 1536 well plate followed by 1 µL of CTG. After 15 minutes of incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

RESULTS

The liver cancer cell line Hep-G2 is an adherent cell line with epithelial morphology that was resuspended from cryopreserved stocks. The ability to transfer cells from cryopreserved stock allows for higher throughput and consistency of cellular source material while minimizing the burden of maintaining lines in culture. Transferring Hep-G2 cells in 10% Histodenz allows for accurate and precise transfer of cells.

Hep-G2 Cell Dispensecells/wellFIGURE 6 u

Hep-G2 frozen stock cell dispense with 0% and 10% Histodenz.

TEC-D107-1.0 7

THP-1 Cells, Suspension Line, Dispensed from Frozen Stock CASE STUDY 3

• Echo Qualified Reservoir (Labcyte # ER-0050)

• THP-1 Cell Line (ATCC-TIB-202)

• Growth Media (RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep)

• Histodenz™ (Sigma # D2158)

• CellTiter-Glo Viability Assay (Promega #G7570)

• 1536 Well TC-Treated Plate (Corning #3726)

MATERIALS

METHODS

THP-1 cells were maintained in growth media in log phase prior to cryopreservation. Cells were thawed at 37oC and resuspended in media, centrifuged into a pellet, and resuspended in growth media at 2x final concentration. Histodenz was dissolved in growth media at 2x final percentage. The cells and Histodenz solutions were combined to create the cell dispense solution and added to the reservoir. The Echo 525 Liquid Handler transferred 1 µL of cell solution to a 1536 well plate followed by 1 µL of CTG. After 15 minutes of incubation, the luminescent signal was read on the BMG LABTECH PHERAstar.

RESULTS

The contact-less transfer does not stress cells the way traditional methods do. Transferring THP-1 cells, thawed from cryopreservation and resuspended in 10% Histodenz, allows for accurate and precise transfer of cells.

FIGURE 7 u

THP-1 frozen stock cell dispense with 0% and 10% Histodenz.

LABCYTE INC.

THP-1 Cell Dispensecells/well

LABCYTE INC.1190 Borregas AvenueSunnyvale, CA 94089, USA

Toll-free: +1 877 742-6548Fax: +1 408 747-2010

SALESNorth America | +1 408 747-2000 | info-us@labcyte.comEurope | +353 1 6791464 | info-europe@labcyte.comJapan | +61 39018 5780 | info-japan@labcyte.comAsia Pacifi c | +61 39018 5780 | info-asiapacifi c@labcyte.comOther | +1 408 747-2000 | info-us@labcyte.com

© 2016 Labcyte Inc. All rights reserved. Labcyte®, Echo®, MicroClime®, the Labcyte logo, and Access™ are registered trademarks or trademarks of Labcyte Inc., in the U.S. and/or other countries.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

All product names and brands are properties of their respective owners.

TEC-D107-1.0AUG 2016

DISCUSSION

As these three case studies demonstrate, the Echo Liquid Handler allows for high-quality cell seeding across a range of cell densities and transfer volumes, even showing potential for single-cell applications. With the use of 1536-well plates and minuscule reaction volumes of just 2 µL, the Echo Liquid Handler reduces input requirements and detection costs, while the Echo Reservoir minimizes dead volume. The protocols described here can be applied to other cell lines of interest.

We are grateful to Elina Huovari, Laura Turunen, and Jani Saarela from the Finnish Institute of Molecular Medicine for their support and developmental work in these experiments.

TECHNICAL NOTE High-Precision Cell Dispensing with the Labcyte Echo® Liquid Handler