SYTOX® AADvancedTM RedCellEventTM 3/7 Green

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Flow Cytometry Multiplexing and its Use in Detailed Characterization of Cell Health

Jeff Haskins, Michael O’Grady, Michelle Yan, Marcy Wickett & Leticia MontoyaThermo Fisher Scientific, Molecular Probes, Eugene, OR, USA

Figure 1a. Varioskan™ High Throughput Screening of Ramos Cells with PrestoBlue Cell Viability Reagent

Cells were plated at 40,000 cells per well and treated with 10 µM of each drug from the KillerCollection from MicroSource Discovery Systems, Inc. and cultured under standard conditionsand either at hypoxic (1% O2) or hyperoxic (19% O2) levels using a Heracell™ VIOS 160i Tri-Gas Incubator. Cells were incubated at three time periods with the compound library. At 24,48, and 72 hours post compound treatment, cells were assayed with either PrestoBlue CellViability Reagent or LIVE/DEAD Fixable Aqua Dead Cell Stain. These were analyzed witheither the VarioskanTM Flash Multimode Reader or the AttuneTM NxT Acoustic FocusingCytometer.

© 2017 Thermo Fisher Scientific Inc. All rights reserved. All Thermo Fisher Scientific and its subsidiaries unless otherwise specified Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com. For research use only. Not for use in diagnostic procedures.

Figure 2. Gambogic acid is more potent than the amide form

ABSTRACTHigh throughput screening (HTS) is an extremely effective method for allowingresearchers to identify putative compounds of interest which play a role in theirarea of cellular research. Recently, flow cytometry has emerged as a powerfulHTS tool with the added benefit of cell-by-cell analysis. Flow cytometry not onlyallows a researcher to study drug effectiveness towards different cell types butalso can be used to analyze protein-protein interactions, metabolic activities, aswell as DNA/mRNA content in a single or multiplexed assay format.In this study, we screened the MicroSource Discovery System Killer Platecompound library and compared HTS using a plate reader with flow cytometermultiplexing. Jurkat and Ramos cells, T and B lymphocyte cell typesrespectively, were used as cell models and cultured under standard conditionsand screened either at hypoxic (1% ) or hyperoxic (19%) oxygen levels at variedlengths of time (24, 48, or 72 hrs). To assess compound veracity, post-screeninganalysis was implemented to establish EC50s of “hits” from the compoundlibrary. Membrane integrity and metabolic activity were measured as an initialscreening output for evaluating cellular viability. Using the multiplexedcapabilities of flow cytometry, we performed secondary and tertiary assays tofurther characterize the possible mechanism of action by analysis of cell cycle,specific RNA expression levels, oxidative stress, and caspase activation acrossdiffering concentrations of the compounds. This method and analysis highlightsthe complicated nature of assessing toxicity in cellular screening assays and theadvantages of Flow Cytometry in particular for characterizing cell health.

RESULTSScreening

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 21

1 0

1 0 0

R a m o s 2 4 h o u r sP re s to B lu e

S a m p le #

RF

U 2 . 7 .

1 0 1 .1 1 0 . 1 7 5 .

2 .

7 . 1 0 1 .

1 1 0 .

1 7 5 .

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 21

1 0

1 0 0

R a m o s 2 4 h o u r sP re s to B lu e

S a m p le #

RF

U 2 . 7 .

1 0 1 .1 1 0 . 1 7 5 .

2 .

7 . 1 0 1 .

1 1 0 .

1 7 5 .

1% O219% O2

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 21

1 0

1 0 0

R a m o s 2 4 h o u r sF ix a b le L iv e D e a d A q u a

S a m p le #

% D

ea

d C

ell

s

2 .

7 .1 0 1 .

1 1 0 .

1 7 5 .

2 .7 .

1 0 1 .

1 1 0 .

1 7 5 .

Selected hits

Figure 3. Flow allows for detailed characterization of the cell cycle

Figure 4. Caspase Assay Confirms Differential Potency of Gambogic Acid

Apoptotic Cells

G0/G1G2/M

S

Apoptotic Cells

G0/G1G2/M

SControl 6 µg GA

FxCycleTM Violet

Clic

k-it®

EdU

AF6

47

Control 6 µg GA

FxCycleTM Violet

Cou

nt

Figure 5. PrimeFlowTM RNA allows for detailed characterization of RNA and Protein Levels

High fluorescenceLow fluorescence

PrestoBlue® Cell Viability ReagentCell viability as indicated by the fluorescence of reduction product. • Resazurin (PrestoBlue reagent) is reduced by the intracellular environment of

living cells. ~Z’ = 0.7• Cell viability was determined by measuring the fluorescence intensity of total

cells per well using a Varioskan™ Flash plate reader.

LIVE/DEAD® Fixable Aqua Dead Cell Stain Cell viability evaluated based on cell membrane integrity.• For cells with compromised plasma membranes, amine reactive dye labels

both the cell surface and the cell interior. For intact cells, dye labels only the cell surface.

• Cell viability was determined by the mean fluorescent intensity (MFI) of total cell population. ~Z’ = 0.8

Figure 1b. AttuneTM NxT High Throughout Screening of Ramos Cells with LIVE/DEAD Fixable Aqua Dead Cell Stain

AttuneTM NxT Autosampler HTS: “Cherry Picking”: Different Readouts Confirm Potency of Drug

0 .0 1 0 .1 1 1 0 1 0 00

5 0

1 0 0

1 5 0

A m s a c r in e # 1 1 0(F ix a b le L iv e D e a d A q u a )

A m s a c rin e (µ M )

No

rma

lized

MF

I

J u rk a t O 2 1 9 %

J u rk a t O 2 1 %

R a m os O 2 1 9 %

R a m o s O 2 1 %

0 .0 1 0 .1 1 1 0 1 0 00

5 0

1 0 0

1 5 0

G a m b o g ic A c id A m id e # 7(F ix a b le L iv e D e a d A q u a )

G a m b o g ic A c id A m id e (µ M )

No

rma

lized

MF

I J u rk a t O 2 1 %R a m o s O 2 1 9 %R a m o s O 2 1 %

J u rk a t O 2 1 9 %

0 .0 1 0 .1 1 1 0 1 0 00

5 0

1 0 0

1 5 0

G a m b o g ic A c id A m id e # 7(P re s to B lu e )

G a m b o g ic A c id A m id e (µ M )

No

rma

lize

d R

FU

J u rk a t O 2 1 9 %

J u rk a t O 2 1 %

R a m o s O 2 1 9 %

R a m o s O 2 1 %

0 .0 1 0 .1 1 1 0 1 0 00

5 0

1 0 0

1 5 0

A m s a c r in e # 1 1 0(F ix a b le L iv e D e a d A q u a )

A m s a c rin e (µ M )

No

rma

lized

MF

I

J u rk a t O 2 1 9 %

J u rk a t O 2 1 %

R a m os O 2 1 9 %

R a m o s O 2 1 %

0 .0 1 0 .1 1 1 0 1 0 00

5 0

1 0 0

1 5 0

G a m b o g ic A c id # 2(F ix a b le L iv e D e a d A q u a )

G a m b o g ic A c id (µ M )

No

rma

lized

MF

I J u rk a t O 2 1 %

R a m o s O 2 1 9 %

R a m o s O 2 1 %

J u rk a t O 2 1 9 %

0 .0 1 0 .1 1 1 0 1 0 00

5 0

1 0 0

1 5 0

G a m b o g ic A c id # 2(P re s to B lu e )

G a m b o g ic A c id (µ M )

No

rma

lize

d R

FU

J u rk a t O 2 1 9 %

J u rk a t O 2 1 %

R a m o s O 2 1 9 %

R a m o s O 2 1 %

Gambogic Acid Response EC50PrestoBlue LIVE/DEAD

19% O2 1% O2 19% O2 1% O20.6 µM 0.6 µM Jurkat ~0.6 µM ~0.5 µM0.2 µM 0.2 µM Ramos ~0.3 µM ~0.3 µM

Gambogic Acid Amide Response EC50PrestoBlue LIVE/DEAD

19% O2 1% O2 19% O2 1% O21.3 µM 1.5 µM Jurkat 2.5 µM 2.8 µM0.5 µM 0.4 µM Ramos 1.0 µM 2.3 µM

Secondary Assays: Test mechanism of Action

Gambogic Acid Response EC50CellEvent 3/7 Green19% O2 1% O2

Jurkat 2.1 µM 1.7 µMRamos 0.8 µM 0.6 µM

FMO Hif1α

FMOHif1α

Hif1 alpha Protein Hif1 alpha PrimeFlow RNA

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SUMMARY• Jurkat and Ramos cells displayed differential responses to compound

treatment when studied across cell health, cell cycle and RNA expressionassays.

• Cell health determination based on reduction potential of the cell using anHTS plate reader format and membrane integrity assay using flowcytometry showed higher potency of Gambogic Acid than it’s derivativecompound Gambogic Acid Amide.

• The statistics of flow cytometry allow for more detailed characterization of“hits”

• Biological applications on flow cytometry (Click-it EdU, Fixable LIVE/DEAD,PrimeFlow RNA, etc…) allow for a more specific analysis of a compound’spossible mechanism of action.

• Future directions- Further analysis of “hits” or lead compounds to includeassays shown here and an expanded miRNA panel.

• Dysfunctional expression of microRNAs is a common feature in leukemiadiseases, comparing the expression profiles of Burkitt’s lymphoma (B-cells)and T-cell acute lymphoblastic leukemia (T-cells) cells , microRNA 342-3pshows higher expression levels in B-cells (Ramos-) than in T-cells (Jurkat).

• Introducing hypoxic conditions to T- and B-cells demonstrates microRNAlevels are diverse. We show that common mciroRNAs such as let-7a areup-regulated in hypoxic conditions (1% O2).

• Human RPL13a is a housekeeping gene that is shown to stay consistentacross the two oxygen levels tested.

• Click-it® EdU allows forfull cell cycle analysis onthe Attune NxT flowcytometer.

• A greater number ofproliferating cells isobserved at 1% vs 19%oxygen levels.

• CellEventTM Caspase-3/7 Green gives characteristics of lysed-, apoptotic-,and normal cells.

• Hif1α is a tumor suppressor gene and is up-regulated in hypoxic conditions.The duration of hypoxia incubation may promote or inhibit leukemiaprogression and maintenance, thus explaining the oncogenic or tumorsuppressor activity.

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