Nick J Dolman*, Kevin M Chambers†, Scott Clarke†, Chris L Langsdorf†, Brian J Agnew†, Daniel W Beacham†, Victoria Robinson †, Bhaskar Mandavilli†, Michelle Yan†,

Richik N Ghosh* and Michael S Janes†

*Thermo Fisher Scientific. 100 Technology Drive, Pittsburgh, PA 15219 USA. †Thermo Fisher Scientific. 29851 Willow Creek Road, Eugene, OR 97402 USA

ABSTRACT Measuring and understanding the health of a population of cells is of critical importance in life science research. Not only does this

indicate what, if anything, an experimental manipulation may have done to the health of cells under investigation but it also impacts

translational research. The health of a cell can be thought of as a continuum. At the top level cells are either alive or dead, commonly

known as viability. Directly below viability is the mechanism of death followed by a third level representating pre-lethal indicators of cell

stress. As new details emerge of the pathways and processes that characterize the health of cellular populations, there has been a need

for new tools to report these processes. Incorporation of automated imaging acquisition and analysis has significantly increased the

throughput and objectivity of these measurements. Here we describe the use of multi-parametric approaches to probe cell health using

high content imaging. We will show high content imaging to measure general viability as well as apoptotic cell death mechanisms. We will

also describe high content assays to report pre-lethal indicators of cellular imbalance including mitochondrial health, autophagy and

proliferation. Finally, we will describe novel tools to probe the internalization of therapeutic antibodies via endocytosis as well as endocytic

pathways themselves.

Visualizing the life and death of cells: novel probes and platforms

www.thermofisher.com

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2015 Thermo Fisher Scientific. All rights

reserved. The trademarks mentioned herein are the property of Thermo Fisher Scientific or their respective owners.

INTRODUCTION

LPS + DPI

CONCLUSIONS

• Here we present a suite of tools to monitor changes in the health of a population of cells

• Using high content imaging these tools enable multi-parametric analysis of viability, mechanisms of cell death

as well as pre-lethal indicators

• Site specific antibody labeling using SiteClick™ allows the attachment of therapeutic agents, fluorescent

reporters or both to therapeutic antibodies

Figure 1: Continuum of cell health. Assessing the health of a population of cells can involve a simple assessment of viability: live or

dead. However more in depth analysis can be achieved by looking at the death pathways initiated, and a plethora of pre-lethal indicators

that manifest themselves before any global change in viability is evident. Moreover multi-parametric acquisition allows simultaneous

interrogation of readouts that span the continuum, revealing important information about the progression from a healthy to unhealthy

state. Here we highlight the use of these tools to provide multi-parametric readouts of cell health using fluorescent probes and high

content imaging.

RESULTS

Figure 3: High content assays for apoptosis

Viable

-RNA/Protein quality control

-Polarized mitochondria

-[ATP] high

-ROS low

-Reducing cytoplasm

-Regulated proliferation

Pre lethal

-Loss of protein quality control

-Depolarized mitochondria

-Oxidizing cytoplasm

-[ATP] low

-ROS bursts

-Deregulated proliferation

Non-Viable

-Loss of membrane integrity

Autophagy/DNA repair

Apoptosis

-Caspase 3/7

-DNA Damage

-Removal of DAMPs

Necrosis/Necroptosis

-RIP kinase (PCD)

-DAMPs present

Immunological consequence

-Phagocytosis (‘find me-eat me’)

-Inflammatory response (DAMPs)

HeLa - Valinomycin

-1 0 1 2 30

200

400

600

800

1000 EC50=25.5 M

Log Valinomycin (M)

Cell M

em

bra

ne

Perm

eab

ilit

y

Hoechst

33342

Image-I

T®

DE

AD

Gre

en

TM

Via

bili

ty

sta

in

120 µM 40 µM 13 µM 4.4 µM

LIVE/DEAD® 488/570

Hoechst 33342/Image-iT™ DEAD Green/Click-iT™ PLUS EdU Alexa Fluor™ 647

Hoechst 33342 Image-iT™DEAD Green Click-iT™ PLUS EdU Overlay

3nM

Cam

pto

thecin

10

M

Cam

pto

thecin

Ve

hic

le

10

uM

Nic

losa

mid

e

BAX-BAK

channels

BID

BAD, BID, HRK

PUMA, NOXA

BIM, BIK,BMF

tBID

Casp8

Casp7

Casp2

Casp3

Casp6

Casp8

Casp10

Casp9

Bcl-2

Granzyme B FADD

Death receptors

Granzyme B

pathway

Extrinsic

pathway

Intrinsic

pathway

CytC

Bcl-2

BH3-only

family

Hoechst33342 TMRM

10nM

CC

CP

1

0

M C

CC

P

Dy Caspase 3/7

CCCP Staurosporine

- + - + - + - +

Mean A

vg

Inte

nsity *** ***

*** **

**

Vehicle 100nM Nocodazole

Compound addition

***

Scramble KIF11

Gene knockdown

1nM 1hr

Log [Bafilomycin A1] M

B C D E

Vehicle

Dynasore

Pitstop 2™

Me

an

Ale

xa

Flu

or®

647

positiv

e p

un

cta

per

ce

ll

Vehicle Dynasore Pitstop 2™

1.0

10

-09

1.0

10

-08

1.0

10

-07

1.0

10

-06

1.0

10

-05

1.0

10

-04

1.0

1.5

2.0

2.5

Me

an

sp

ot in

ten

sity

Log [Dynasore] uM

pHrodo™ 10k dextran

MD

A-M

B-2

31

S

K-B

R-3

Alexa Fluor® 647 MMAE

Herceptin®

MMAE

Herceptin®

A p o p to s is In d u c tio n (C e llE v e n t™ C a s p a s e -3 /7 G re e n )

Co

ntr

ol

0.0

0000063

0.0

000025

0.0

0001

0.0

004

0.0

016

0.0

063

0.0

25

0.1

0

2 0

4 0

6 0

8 0

1 0 0

S K -B R -3 (H E R 2 + )

M D A -M B -2 3 1 (H E R 2 -)

A n tib o d y C o n c e n tra tio n ( g /m L )

% A

po

pto

tic

Ce

lls

A le x a F lu o r

6 4 7 In te rn a liz a tio n

Co

ntr

ol

0.0

0000063

0.0

000025

0.0

0001

0.0

004

0.0

016

0.0

063

0.0

25

0.1

0

2 0 0

4 0 0

6 0 0

8 0 0

S K -B R -3 (H E R 2 + )

M D A -M B -2 3 1 (H E R 2 -)

A n tib o d y C o n c e n tra tio n ( g /m L )

Inte

ns

ity

(R

FU

)

H+

e-

H2O

H+

ATP

Matrix

Inter membrane space

Dy

Figure 1: Continuum of cell health.

Figure 2: High content imaging of viability.

3n

M C

am

pto

thecin

1

0

M C

am

pto

the

cin

A B

C

D E

A B C D

A B C

A B

C

A C

B

A B C

Figure 2: High content imaging of viability. A. Delineation of cellular viability with Image-iT™ DEAD green. The integrity of the plasma

membrane is lost in late stage cell death, Image™ DEAD Green is only able to stain cells without an intact plasma membrane. B.

Valinomycin causes a dose-dependent increase in cell death as reflected by an increase in Image-iT™ DEAD Green staining (top),

representative images (bottom). Hoechst 33342 is used to count total cells. Automated image acquisition and analysis was performed on

the Thermo Scientific ArrayScan™ VTI. C. Combined viability and proliferation using Image-iT™ DEAD green and Click-iT™ PLUS EdU

Alexa Fluor™ 647. Treatment of A549 cells with 10M camptothecin causes a concurrent increase in Image-iT™ DEAD Green staining

and a decrease in EdU incorporation compared to treatment with low (3nM) concentrations of Camptothecin. Automated image acquisition

wand analysis was performed on the Thermo Scientific CellInsight™ Cx5 High content Screening (HCS) Platform. D. Two-color viability

assays using the LIVE/DEAD cell imaging kit. Live cells retain Live Green probe but aren’t permeable to the Dead Red Probe. During cell

death the plasma membrane is compromised. Live green is no longer retained whereas Dead red is now able to gain access E.

Demonstration of two-color viability assay in high-content imaging. Dose-dependent cell death induced by Camptothecin in A549 cells.

With increasing concentrations of camptothecin the Live Green stain is lost as the plasma membrane is compromised, there is a

concomitant increase in Dead Red staining as the impermeant dye can now gain access to cells where the plasma membrane has been

permeabilized (left). Representative images (right). Automated image acquisition and analysis was performed on the Thermo Scientific

CellInsight™ Cx5 High content Screening (HCS) Platform F. HeLa spheroids labeled with LIVE/DEAD Cell Imaging kit (488/570.

compared to vehicle treated cells those treated with Niclosamide show increased death as shown by decreased Live Green staining and

increased Dead Red staining. Spheroids were imaged with a laser-scanning confocal microscope.

Figure 3: High content assays for apoptosis. A. Pathways leading to the induction of apoptosis. B.

U-2 OS cells were treated with varying dose of staurosporine for 24 hours. Cells were then labeled with

the fluorogenic caspase sensor CellEvent™ Caspase-3/7 Green Detection Reagent. Cells were co

stained with Hoechst 33342 stain and imaged on the Thermo Scientific ArrayScan™ VTI. A dose-dependent

increase in the percent of cells positive for active Caspase 3/7 was observed with increasing concentrations of

staurosporine C. HeLa cells were treated with 0.5 μM staurosporine in the presence of 0– 30 μM Caspase 3/7

Inhibitor 1 (EMD Chemicals) for 4 hours. Cells were then labeled with CellEvent™ Caspase-3/7 Green Detection

Reagent and Hoechst 33342 stain. Automated image acquisition and analysis was performed on the Thermo

Scientific ArrayScan™ VTI. A dose-dependent decrease in the percent of cells positive for active Caspase 3/7 was

observed when cells were incubated in the presence of Caspase 3/7 inhibitor 1, indicating specificity of the probe

for active caspase 3/7 D. U2OS cells were treated with 30 µM etoposide for 48 hours or vehicle control and labeled

with 7.5 µM CellEvent™ Caspase-3/7 Green Detection Reagent for 30 minutes followed by Hoechst 33342 stain.

Cells were analyzed on a Thermo Scientific Arrayscan™ VTI, and percent of cells positive for active caspase 3/7

was determined.

Figure 4: High content assays for mitochondrial membrane potential

D E

Figure 4: High content assays for mitochondrial membrane potential. A. Establishment of mitochondrial membrane potential and the

production of ATP B. HeLa cells were loaded with 50nM TMRM and treated with varying doses of CCCP for one hour. There is a dose-

dependent decrease in TMRM staining reflecting a loss of mitochondrial membrane potential. Images acquired using a Thermo Scientific

Cellomics™ ArrayScan™ VTI. C. HeLa cells were loaded with 50 nM TMRM (red) followed by 5 µM CellEvent Caspase 3/7 Substrate

(green). Cells were then treated with 0.5 µM staurosporine or 100nM CCCP. Automated image acquisition and analysis was performed on the

Thermo Scientific ArrayScan™ VTI. D. HeLa cells were treated with troglitazone and assayed using the fixable Mitochondrial Health Kit and

show a loss of both plasma membrane integrity and mitochondrial membrane potential at higher concentrations of troglitazone. E. HeLa cells

treated with varying concentrations of troglitazone, incubated for 24 hours and assayed using the HCS Mitochondrial Health Kit. EC50 values

were calculated from the dose response curves with respect to mitochondrial membrane potential (left) and plasma membrane integrity

(right). Automated image acquisition and analysis was performed on the Thermo Scientific ArrayScan™ VTI.

Figure 5: High content assays for proliferation

Figure 5: High content assays for proliferation. A Basis of proliferation using EdU (5-ethynyl-2'-deoxyuridine) versus immunodetection of

BrdU. Both BrdU and EdU are incorporated by cells synthesizing DNA. EdU is detected through a copper catalysed cycloaddition (Click

reaction) between the alkyne in EdU and a azido modified fluorescent dye. In contrast BrdU requires significantly longer protocols needed

for anti-BrdU. EdU B. A549 cells were either incubated with 100nM nocodazole for 24 hours (left) or KIF11 knocked down using siRNA

(right) for 48 hours. Cells were incubated with EdU for one hour and then fixed and permeabilised and labeled with Click-iT™ PLUS EdU

Alexa Fluor™ 647. A significant reduction was in EdU incorporation under conditions of mitotic block, caused either by drug treatment of

gene knockdown. Automated image acquisition and analysis was performed on the Thermo Scientific CellInsight™ Cx5 High content

Screening (HCS) Platform. C. A549 cells were treated overnight with varying does of taxol. The following day cells were incubated with EdU

and then fixed and permeabilised before detection of the EdU with the Click-iT™ PLUS EdU Alexa Fluor™ 647 imaging kit. Taxol causes a

dose-dependent inhibition of EdU incorporation. Data acquired on the Thermo Scientific CellInsight™ Cx7 High content Screening (HCS)

Platform

Figure 6: High content assays for autophagy

A

C D

Figure 6: High content assays for autophagy. A. Formation of the autophagosome, recruitment of LC3B and subsequent fusion with the

lysosome. B. Stimulating autophagy through mTOR inhibition (10M PP242, 30 mins) caused a significant increase in the intensity of LC3B

positive puncta in IBMK cells stably expressing GFP-LC3B. Automated image acquisition and analysis was performed on the CellInsight™

Cx5 High content Screening (HCS) Platform C. Correlation of LC3B spot intensity and LC3B spot number under bafilomycin A1 mediated

block of autophagy in IBMK cells stably expressing GFP-LC3B co-stained with Hoechst 33342. Automated image acquisition and analysis

was performed on the Thermo Scientific CellInsight™ Cx5 High content Screening (HCS) Platform D. Dose-dependent accumulation of

autophagosomes by 5 different compounds. A549s were treated with concentration ranges of the stated compounds overnight. Cells were

fixed & LC3B detected with rabbit anti-LC3B IgG (0.5g/ml) and an Alexa Fluor™ 647 dye-conjugated goat anti-rabbit secondary. Nuclei were

stained with Hoechst 33342. Automated image acquisition and analysis was performed on the Thermo Scientific ArrayScan® VTI.

Figure 7: High content assays for endocytosis.

Figure 7: High content assays for endocytosis. A. Routes of endocytosis, recycling and trafficking to intracellular compartments B.

A549 cells were pre-treated with Vehicle (DMSO) 100M Dynasore or 50M Pitstop 2™ for 16 hours under regular culture conditions.

Cells were then incubated with 100g/mL dextran, Alexa Fluor™ 647, 10000 mw, anionic, fixable in HBSS supplemented with 20mM

HEPES (pH 7.4) for one hour at 37oC. Representative images of A549 cells pretreated with vehicle or compound then incubated with

100ug/mL dextran, Alexa Fluor™ 647, 10000 mw, anionic, fixable. Cells were counter stained with Hoechst 33342 (Blue). C. HeLa cells

were pre-treated with a concentration range of Dynasore for 2 hours under regular culture conditions. Cells were then incubated with

40g/mL dextran, pHrodo™, 10000 mw, in HBSS supplemented with 20mM HEPES (pH 7.4) for one hour at 37oC. Cells were washed

in dye free HBSS, counter stained with Hoechst 33342. Automated image acquisition and analysis was performed on the Thermo

Scientific ArrayScan™ VTI.

Figure 8: High content assays for therapeutic antibody uptake and killing. A Site specific antibody labeling using SiteClick™ labeling

system. This system allows efficient site-selective attachment of one or multiple fluorescent dyes, radiometal chelators, or small-molecule

drugs to antibodies. B HER2+ SK-BR-3 cells and HER2- MDA-MB-231 cells were treated with varying concentrations of dual SiteClick™

Herceptin® Alexa Fluor™ 647/MMAE conjugates for 72 hours. Cells were then labeled with CellEvent™ Caspase-3/7 Green detection

reagent. Automated image acquisition and analysis was performed on the Thermo Scientific ArrayScan™ VTI. SK-BR-3 cells internalized

significant amounts of antibody-drug conjugate at higher concentrations, while MDA-MB-231 cells had minimal internalization (left). SK-BR-3

cells had dramatic cell death at higher ADC concentrations, while MDA-MB-231 cells had consistently low levels of apoptosis (right). C.

Representative images from HER2+ SK-BR-3 cells and HER2- MDA-MB-231 treated with MMAE Herceptin (+/- Alexa Fluor™ 647

conjugation) counter stained with Hoechst 33342

Figure 8: High content assays for therapeutic antibody uptake and killing.

LC3B

Vh mTOR inhibition

Vh

mTOR inhibition

B

F

Lysosome

Isolation membrane Autophagosome Autophagolysosome

Hoechst 33342

Image-iT™ DEAD Green

Viability stain

MitoHealth Stain

1nM Taxol 1M Taxol