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Version 1.0 Dec, 2019
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Version 1.0 Dec, 2019
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Content
Chapter 1 Overview 2
1.1 Intended Use 2
1.2 Description 2
1.3 Workflow 3
Chapter 2 Required Materials and Equipments 4
Chapter 3 Reagent Preparation 6
Chapter 4 Blood Collection and Handling 7
4.1 Phlebotomy 7
4.2 Transportation 7
Chapter 5 Leucosep Preparation 8
5.1 Pre-Blocking of Tips or Tubes 8
5.2 Leucosep Preparation 8
Chapter 6 RBC Lysis 10
Chapter 7 WBC Depletion 11
Chapter 8 CSV Enrichment 13
Chapter 9 Cell Immunostaining 15
Chapter 10 Microscope Examination 16
Chapter 11 Maintenance 17
11.1 Histopaque® Troubleshooting 17
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Overview
1.1 Intended Use
LiquidCell™ is intended for separating circulating rare cells from
human bodily fluids utilizing functionalized magnetic beads and a
high-performance glass slide for maximal cell capture and retention.
LiquidCell™ is designed for life science and clinical research
applications supported by wide repertoire of pre-validated,
standardized GMP bioregeants targeting cancers and clinical
relevant biomarkers.
1.2 Description
LiquidCell™ is an open platform for manual processing of red
blood cell lysis, white blood cell depletion via antibody conjugated
magnetic beads, and centrifugal cytospin to capture and retain all
circulating rare cells onto a specialty-coated, glass slide for
downstream immunofluorescent and molecular staining, cell
picking, and next-generation sequencing. The glass slide is
compatible with commercial automated immuno- and in situ
hybridization stainers for automated, high-throughput
processing. Moreover, LiquidCell™ is amenable to
tyramide-based fluorescent amplification and non-fluorescent
chromogenic staining. Cross-contamination is minimized by
adopting single-use consumables.
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1.3 Workflow
Blood Collection
Leucosep Preparation
(Operating time: ~30 min)
RBC Lysis (Operating time: ~10 min)
WBC Depletion (Operating time: ~40 min)
CSV enrichment (Operating time: ~60 min)
Cell Immunostaining (Operating time: ~130 min)
Microscope Examination
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Required Materials and Equipments
Depletion Kits Catalog# Brand
CD45 Depletion Kit CSV enrichment antibody.
Kit content:
1. CD45 Magnetic AbBeads
2. RBC Lysis Buffer
3. CTC Buffer 1
4. CTC Buffer 2
5. Mouse Anti-CSV monoclonal antibody
DM0008 Abnova
3D Magnetic Separator DU0009 Abnova
Specialty-Coated Glass Slide Catalog# Brand
SuperSlide™ DL0001 Abnova
Antibody Catalog# Brand
Cell-Surface Vimentin (CSV) monoclonal antibody (FITC) DH0043 Abnova
CD45 monoclonal antibody (PE) DH0044 Abnova
CD45 monoclonal antibody PLUS (PE) DH0090 Abnova
Reagent Catalog# Brand
FcR Blocking Reagent U0318 Abnova
Staining Blocker U0330 Abnova
50X Antibody Dilution Buffer (50X ADB) U0320 Abnova
Leukocyte Aggregation Inhibitor DU0008 Abnova
Hoechst 33342 U0334 Abnova
Anti-Mouse IgG MicroBeads 130-048-402 Miltenyi Biotec
Chemical Catalog# Brand
8% Paraformaldehyde (formaldehyde) Aqueous Solution 157-8 Electron Microscopy
Science
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UltraPure™ 0.5M EDTA, pH 8.0 15575020 Fisher Scientific
DPBS powder 21600010 Gibco
Consumable Catalog# Brand
Bovine Serum Albumin lyophilized powder A2153 Sigma-Aldrich
LS Columns 130-042-401 Miltenyi Biotec
50 mL Centrifuge Tubes 352070 Fisher Scientific
K2-EDTA Blood Collection Tube 367525 BD Biosciences
Eppendorf Micro Test Tubes 0030 121.589 Eppendorf
Histopaque®-1077 10771 Sigma-Aldrich
Leucosep® 227290 Greiner Bio-One
Cyto Funnel, 8 mL M966-8 Simport CANADA
Cover slide 0101040 Marienfeld-Superior
Equipments Catalog# Brand
Hettich Centrifuge university 320 R 1406 Hettich
Cyto rotor, 6-place 1626 Hettich
Carriers 1660 Hettich
MACS MultiStand 130-042-303 Miltenyi Biotec
Dako pen #S200230 Agilent,Dako
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Reagent Preparation
All the reagents/solutions used should be of molecular grade and
sterile-filtered!
4% PFA (4% Paraformaldehyde Aqueous Solution)
Dilute 8% paraformaldehyde aqueous solution with 1X PBS. 4%
PFA can be stored at 4°C for one week.
1X ADB
Dilute 50X ADB by adding adequate 1X PBS to 50X ADB. 1X ADB
should be kept on ice or at 4°C and used within 2 days.
MACS buffer
Prepare a solution containing PBS (pH 7.2), 0.5% bovine serum
albumin (BSA), and 2 mM EDTA
10% Glycerol/PBS
1. Dilute 1 mL of glycerol in 9 mL of PBS.
2. Sterilize the solution by passing it through a 0.22-μm filter.
3. Store in 500-μL aliquots at 4°C
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Blood Collection and Handling
4.1 Phlebotomy
Access to a peripheral vein should be performed with a 22G needle.
The first 2 mL of collected blood should be discarded to avoid
potential skin cell contamination from venepuncture. Then transfer
10 mL of blood sample in each K2-EDTA Blood Collection Tube.
Gently rotate the 10 mL blood tube end-over-end five times
immediately. Place the collected blood sample at room temperature
before use.
Do not use blood collection tubes containing fixatives to avoid
permeabilization of cell membranes.
4.2 Transportation
Blood has a maximum preservation time of 12 hours at room
temperature. For example, if the blood is drawn at 9 am in a hospital,
it should be processed before 9 pm.
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Leucosep Preparation
5.1 Pre-Blocking of Tips or Tubes
Rinse tips, pipets and tubes with CTC Buffer 1 once before use.
5.2 Leucosep Preparation
Wrap the Histopaque® -1077 stock with aluminum foil and
store at 4℃ in the dark before use. Refer to Histopaque®
Troubleshooting in Chapter 10.
Bring refrigerated 40 mL PBS to room temperature (RT)
overnight before the day of the experiment. Keep the PBS in
RT until it is used up.
5.2.1 Add 3mL Histopaque® -1077 into 15 mL Leucosep® tube
(Tube A1). Repeatly, add 3 mL Histopaque® -1077 into 15
mL Leucosep® tube (Tube A2).
5.2.2 Spin down to make Histopaque® -1077 stay in the tube
bottom, briefly
5.2.3 Dilute 7.5 mL blood sample at 1:1 ratio with 1X PBS in
Falcon® 15 mL Centrifuge Tube by using a Pipette Aid.
Invert the tube gently. The final volume is 15 mL.
5.2.4 Carefully add 7.5 mL of diluted blood sample into Tube A1
and Tube A2.
5.2.5 Centrifuge Tube A1 and A2 at 1000 xg in roomtemperture
for 10 minutes.
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After centrifugation in 5.2.5, do not proceed further if the density
gradient color and layer in the Leucosep® tube resemble the
examples below due to preexisting low density RBC or RBC
lysis in the patient’s blood sample. Low density RBC and RBC
lysis will adversely affect downstream CTC enrichment and
recovery.
5.2.6 Discard the supernatant above 5 mm PBMC layer with
suction, then transfer the PBMC layer into 50 mL tube
( Tube B, pre-blocking) with broad tip (pre-blocking)
carefully.
5.2.7 Wash Tube A1 and Tube A2 with 3 mL CTC Buffer 1, then
transfer the wash buffer to into Tube B.
5.2.8 Add CTC Buffer 1 to Tube B until 45 mL, centrifuge 300g, 5
min, discard supernatant.
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RBC Lysis
6.1 Resuspension in 1 mL CTC Buffer 1 (make sure all cell pellet
suspense in the buffer completely), add 9 mL RBC Lysis Buffer
gently, stay at RT for 3 min.
6.2 Add CTC Buffer 1 to final 45 mL, centrifuge 300g, 5 min, discard
supernatant.
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WBC Depletion
7.1 Re-suspend cells and add 2 mL CTC Buffer 1 and 4 μL
Leukocyte Aggregation Inhibitor (make sure cell pellet suspense
in buffer completely), then add 1 mL CD45 Magnetic AbBeads
(from 125 μL CD45 Magnetic AbBeads that is pre-blocked with 1
mL CTC Buffer 1 for 30 min), shaking at 125 rpm, RT, for 15 min.
7.2 Place Tube B on 3D Magnetic Separator for 2 min, transfer all
Sup. above the beads into 50 mL tube (Tube C, pre-blocking)
with 1 mL tip (pre-blocking).
***Keep tip for later usage (step 7.3 ~ 7.5.)
7.3 Re-suspend beads in 1 mL MACS Buffer by pipetting with 1 mL
tip (pre-blocking, from step 7.2) for 5 times.
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7.4 Place Tube B on 3D Magnetic Separator for 2 min, transfer all
Sup. above the beads with 1 mL tip (pre-blocking, from step 7.2)
into 50 mL tube (Tube C).
7.5 Repeat step 7.3 ~ 7.4 once.
7.6 Centrifuge 300g for 5 min, discard Sup. till approximately 0.1 mL.
7.7 Place Tube C on 3D Magnetic Separator for 2 min to remove
beads completely.
7.8 Transfer 100 μL supernatant into 15mL tube.
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CSV Enrichment
8.1 Add mouse anti-CSV mAb to 40 μg/mL and incubate at 4℃ for
20min (tap halfway).
8.2 Add 3 mL MACS buffer and centrifuge 300g for 10min. Discard
supernatant.
8.3 Resuspend cell pellet in 100 μL MACS Buffer, add 10 μL
anti-Mouse IgG MicroBeads, mix well and incubate for 20min at
4℃ (tap halfway)
8.4 Add 3 mL MACS buffer and centrifuge 300g for 10min. Discard
supernatant.
8.5 Resuspend cell pellet in 500 μL MACS Buffer
8.6 Place a LS column on MACS MultiStand, add 3ml MACS Buffer
onto the LS column.
8.7 Apply cell suspension onto LS column.
8.8 Wash LS column with 3ml MACS Buffer 3 times.
8.9 Remove LS column on 15ml tube, add 5ml MACS Buffer onto
the LS column, Immediately flush out the magnetically labeled
cells by firmly pushing the plugger into the column.
8.10 Centrifuge 300g for 10min, Discard Sup. till ~ 0.2ml.
8.11 Re-suspense the cell pellet by tapping, then add 10 mL CTC
Buffer 2.
8.12 Centrifuge 300g for 5min, discard Sup. till approximately 400 μL.
8.13 Assembly 2-well SuperSlide.
8.14 Wash 2-well SuperSlide with 1 mL PBS twice.
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8.15 Transfer 200 μL supernatant into 2-well SuperSlide with 200 μL
tip, respectively.
***Keep tip for later usage (step 8.16 ~ 8.17.)
8.16 Wash Tube C with 200 μL of CTC Buffer 2 then transfer to one of
2-well SuperSlide (from step 8.15)
8.17 Wash Tube C with another 200 μL of CTC Buffer 2 then transfer
to another one of 2-well SuperSlide (from step 8.15)
8.18 Stay at RT for 1 hr, add 60 μL of 4% PFA into each well of 2-well
SuperSlide, stay at 4 degree for overnight.
8.19 Add 73 μL of 4% PFA into each well of 2-well SuperSlide, stay at
RT for 15 min.
8.20 Cytospin of 2-well SuperSlide at centrifuge 300g, 5 min by
Hettich Centrifuge university 320 R (loosen the chamber lockers
before centrifuge).
8.21 Remove chamber.
8.22 Wash with PBS twice
8.23 Add 200 μL PBS onto each sample area, immediately.
8.24 Ready for staining.
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Cell Immunostaining
9.1 Circle sample area with DAKO pen.
9.2 Blocking with 150 μL of blocking reagent each well (65 μL 1X
ADB + 60 μL FcR Blocking Reagent + 25 μL Staining Blocker) at
RT for 20 min.
9.3 Remove blocking reagent.
9.4 Add 150 μL of antibody each well (84.3 μL 1X ADB + 25 μL FcR
Blocking Reagent + 25 μL Staining Blocker + 3 μL Cell-Surface
Vimentin (CSV) monoclonal antibody (FITC) + 11.7 μL CD45
monoclonal antibody (PE) + 1 μL CD45 monoclonal antibody
PLUS (PE) in blocking reagent) at RT for 1hr.
9.5 Wash with PBS for 3 times.
9.6 Add 150 μL 10 μg/mL Hoechst 33342 in PBS on slide, stay at RT
for 30 min.
9.7 Wash with PBS for 3 times.
9.8 Add 30 μL 10% glycerol/PBS then cover with cover slide.
9.9 Seal cover slide with nail polish.
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Microscope Examination
10.1 Fluorescent Filters & Exposures
The following microscope filters and exposures have been setup for
Nikon TiE fluorescent microscope to achieve the best staining
results. Each microscope needs be adjusted based on its
specification and staining compatibility. We highly recommend
starting with peripheral blood white blood cells and spike cells to
establish the baseline exposure and staining to avoid false positive
and false negative results.
Fluorochrome EX
(nm)
EM
(nm) Objective Filter Exposure (ms)
FITC 489 515 20X SemRock,
Cat# SpGr
400-800 ms
(no longer than 1 s)
Hoechst 33342 350 451 20X Chroma,
Cat# 49000 < 100 ms
R-phycoerythrin (PE) 565 578 20X SemRock,
Cat# SpOr
400-800 ms
(no longer than 1 s)
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Maintenance
11.1 Histopaque® Troubleshooting
Cause1 Solution
1
Blood draw n >24 hours prior to
separation
Use blood drawn from 2-6 hours prior to separation.
Blood drawn >24 hours will be more difficult to
separate, and percent recovery and viability will be
lower.
Blood has coagulated due to absence
of anticoagulant
Use of vacuum tubes with premeasured amounts of
anticoagulant is suggested. Use of a syringe
(without anticoagulant) is not recommended.
Blood has coagulated due to
incomplete mixing
Ensure vacuum tubes with anticoagulant are mixed
well after blood draw.
Blood sample is too warm and
separation attempted too soon after
blood draw
Red blood cell contamination may be a result of
blood not being at room temperature. After drawing,
the blood should be allowed to cool at room
temperature for minimum 30-45 minutes. If used
immediately after being drawn, the number of
mononuclear cells collected will be low. Separation
procedures are optimal when both blood and
Histopaque are at 18–20 °C, with an acceptable
temperature range of 18–26 °C.
Differences in donor blood physiology
(when single sample has poorer
recovery)
High lipid levels, rheumatoid factor, anemia, and
drug treatment are all possible causes for poor
separation of a specific donor’s blood. If the plasma
is not clear, this is an indication of high lipid levels.
Occlusion of white blood cells in
samples with high levels of white cells
For routine blood specimens, percent recovery will
often be higher for undiluted blood. However,
samples from donors with abnormally high white cell
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counts should be diluted to reduce the size of the
red cell aggregates and improve recovery of white
cells.
Histopaque temperature is too cold
A 100 mL or 500 mL bottle of Histopaque stored at
2–8 °C may take several hours to reach 18–20°C.
When planning to use Histopaque, we recommend
removing the Histopaque from the refrigerator the
previous day and let the bottles stand on the bench
overnight. This ensures the solution is at room
temperature and ready for use.
Incorrect centrifugation force used
The centrifuge should be checked to ensure proper
calibration. Excessive force (higher centrifugation
speed) will lower yield.
Cell pellet does not form single cell
suspension
When resuspending the cell pellet, only a small
amount of wash solution should be used. We
recommend no more than 0.5 mL wash media when
performing the procedure in a 15 mL or 50 mL
centrifuge tube. This wash media is gently rinsed
over the pellet.
Loss of cells during washing due to
adhesion to centrifuge tube wall
Use low protein binding centrifuge and
microcentrifuge tubes.
1. Histopaque® Troubleshooting Guide
https://www.sigmaaldrich.com/technical-documents/articles/biofiles/histopaque-troubleshooting-guide.html
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Abnova (Taiwan) Corporation Neihu Plant 9th Fl., No.112 & No.114, Jhouzih St., Neihu District, Taipei City, 114 Taiwan