Veronica Calderon, Leticia Montoya, Michelle Yan, Dan Beacham, Marcy Wickett, April Anderson, Carolyn DeMarco, and Michael O’Grady
Thermo Fisher Scientific, Willow Creek, Eugene, OR, USA, 97402
ABSTRACT
High throughput screening (HTS) is an extremely effective method for
allowing researchers to identify putative active compounds for therapeutics.
Recently, flow cytometry has emerged as a powerful HTS tool with the added
benefit of cell-by-cell analysis. For these studies, we will be screening a
selection of killer drugs from MicroSource Discovery Systems using flow
cytometry multiplexing. The mechanism of action will be further evaluated
based on killer drug “hits”.
As cell models, Ramos (B-Cell derived) and Jurkat (T-Cell derived) cells were
used at 24, 48, and 72 hrs post-treatment. Membrane integrity and metabolic
activity were measured as an output for evaluating cellular viability. To assess
cell viability, cells were monitored at either normoxic (19%) or hypoxic (1%)
oxygen levels using standard cell culture conditions. To further understand
cellular activity, post-screening analysis was used to establish EC50s of “hits”
from the compound library.
“Hits” identified from the flow cytometry screen were further analyzed to
assess the mechanism of cellular toxicity. To assess cell death, apoptotic
proteins Bcl-2 or CD95 expression was measured. Additionally, expression
of hypoxia-inducible factor-1α; a marker for hypoxia and potential mediator of
inflammation was evaluated post-treatment.
Results indicated that compound “hits” and potency differed in the screen
depending on cell type, the mechanistic readout, length of compound
exposure, and mode of readout used to perform the HTS experiments. The
apoptotic pathway and inflammatory response produced differed based on
cell type, oxygen level, and length of compound exposure. Overall, these
studies will demonstrate the ability of Flow Cytometry-based HTS to rapidly
screen large quantities of compound drugs along with accelerating the
capability to understand their mechanism of action.
INTRODUCTION
High-throughput screening has become the industry standard for pharmaceutical
drug screening and drug analysis. The ideal tool for investigating drug functions on
cells is flow cytometry. Within this study we used an acoustic flow cytometry platform
to analyze cell viability. We further investigated the difference in oxygen levels and
compound potency. The selection of promising compound leads is often poorly
characterized due to the oxygen level difference in clusters of abnormal cell types
within the human body.
Jurkat (T lymphocyte cell type) and Ramos (B lymphocyte cell type) cells were used
to study the affect of drugs on different types of white blood cells. White blood cells
protect the body against foreign intruders; using these cells allows us to evaluate the
effect of drugs tested and their involvement in cell-mediated immunity (T cells) and
humoral immunity (B cells).
Hypoxia-Inducible Factor-1α (HIF-1α), involved in oxygen homeostasis, is activated
by a pro-inflammatory responses and has been shown to play a role in cancer
development1.
Cell death by way of apoptosis can be triggered by either the intrinsic or extrinsic
pathway. The B-cell lymphoma 2 (Bcl-2) family of proteins controls the intrinsic
pathway; also known as the stress or mitochondrial pathway. The extrinsic or death
receptor pathway is controlled by death receptors(DR) signaling, including CD95
(APO1/FAS)2.
MATERIALS AND METHODS
LIVE/DEAD Fixable Aqua Dead Cell Stain – Used to evaluate cell viability
by flow cytometry. Cell viability was determined by the mean fluorescent
intensity (MFI) of total cell population. Reactive dye permeates compromised
cell membranes and reacts with free amines on the cell surface and the
interior on the cell (Cell Membrane Integrity Read-out). ~Z’ = 0.8
PrestoBlue Cell Viability Reagent – Used to measure cytotoxicity and the
proliferation of cells by a spectrometer. Cell viability was determined by
measuring the mean fluorescent intensity of total cells per well and obtaining
the relative fluorescent units (RFU) per well. Resazurin (PrestoBlue reagent)
is reduced by the environment of living cells (Metabolic Read-out). ~Z’ = 0.7
Extracellular antibodies: Hypoxia-Inducible Factor 1α (HIF-1α) AF488
(Novus Biologicals), CD95/FAS APC, Bcl-2 PE
Attune NxT Flow Cytometer – Designed to use acoustic-assisted
hydrodynamic focusing, allowing for fast sample throughput rates.
Varioskan Flash – Spectral scanning reader for fluorescence, absorbance,
time resolved fluorescence, and luminescence.
Flow Cytometry Multiplexing Used to Analyze Metabolic Activity and Assess
Pharmaceutical Compound Toxicity as A High Throughput Screening Tool
Flow Analysis
CONCLUSIONS
Analysis of cell health by screening can benefit from a multi-parametric approach, including
different mechanistic and platform readouts.
Flow cytometry and the use of multi-well plate auto samplers is an excellent tool for high-
throughput screening, and should be considered as a viable option for primary, secondary or
tertiary compound screening.
Jurkat and Ramos cells display differential responses to Amsacrine and Gambogic acid
Future directions: continue tertiary compound screening to investigate mechanisms of
action
For Research Use Only.
Not for use in diagnostic procedures.
© 2016 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
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
R a m o s 2 4 h o u r s
P 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 2
1
1 0
1 0 0
J u r k a t 2 4 h o u r s
P 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 .
Figure 1b. Screening of Ramos and Jurkat Cells Using LIVE/DEAD Fixable Aqua Dead Cell Stain as the
Read-out
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
1
1 0
1 0 0
R a m o s 2 4 h o u r s
F ix a b le L iv e D e a d A q u a
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% D
ea
d C
ells
2 .
7 .
1 0 1 .
1 1 0 .
1 7 5 .
2 .
7 .
1 0 1 .
1 1 0 .
1 7 5 .
Figure 1a. Screening of Ramos and Jurkat Cells Using PrestoBlue Cell Viability Reagent as the Read-
out
Cells plated at 40,000 cells per well, then treated with 10 µM of each drug from the Killer Collection
from MicroSource Discovery Systems, Inc., and stored at either 19% Oxygen or 1% Oxygen in 5%
CO2 at 37 ºC for 24, 48, and 72 hours. At 24, 48, and 72 hours cells were treated with either
LIVE/DEAD Fixable Aqua Dead Cell Stain or PrestoBlue Cell Viability Reagent and analyzed on either
the Attune NxT or the Varioskan Flash.
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1 9 %
1 %
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
C lo fa z im in e # 1 7 5
(F ix a b le L iv e D e a d A q u a )
C lo fa z im in e (µ M )
No
rm
ali
ze
d M
FI
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 %
Clofazimine (antibiotic used in multi-drug
therapy for leprosy) is a more potent drug at
19% Oxygen for both Jurkat and Ramos
cells when analyzed using cellular reduction
potential as a cell health readout.
Figure 3. Oxygen Levels have an Effect on Drug Potency
0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
C lo fa z im in e # 1 7 5
(P re s to B lu e )
C lo fa z im in e (µ M )
No
rm
ali
ze
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 0
0
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
rm
ali
ze
d M
FI
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 %
Clofazimine
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 10.69 µM ~ 30.86 µM NA NA
Ramos 4.77 µM ~ 9.21 µM NA NA
PrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
IC50 Drug Dose Response Table
0 .0 1 0 .1 1 1 0 1 0 0
0
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
rm
ali
ze
d M
FI
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 0
0
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
rm
ali
ze
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 0
0
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
rm
ali
ze
d M
FI 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 0
0
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
rm
ali
ze
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 %
Figure 4. Similar Drugs Have Different Effects on Cell Type
Gambogic Acid (natural product from Garcinia hanburyi shown to activate caspases and have
anti-cancer activity) is a more potent drug than the amide version of same compound.
0 .0 1 0 .1 1 1 0 1 0 0
0
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
rm
ali
ze
d M
FI
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 0
0
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
rm
ali
ze
d M
FI
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 %
Gambogic
Acid Amide
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 0.60 µM 0.58 µM 2.50 µM 2.78 µM
Ramos 0.19 µM 0.19 µM 1.01 µM 2.25 µM
PrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
IC50 Drug Dose Response Table
Screening Cherry Picking
0 .0 1 0 .1 1 1 0 1 0 0
0
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
rm
ali
ze
d M
FI
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 %
Amsacrine (known chemotherapeutic for
Leukemia) is a more potent drug for Jurkat
cells than Ramos cells.
Figure 2. Jurkat and Ramos Cells React Differently to the Same Drug
0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
A m s a c r in e # 1 1 0
(P re s to B lu e )
A m s a c r in e (µ M )
No
rm
ali
ze
d R
FU
J u rk a t O 2 1 9 % A
J u rk a t O 2 1 % A
R a m o s O 2 1 9 % A
R a m o s O 2 1 % A
0 .0 1 0 .1 1 1 0 1 0 0
0
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
rm
ali
ze
d M
FI
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 %
Amsacrine
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat ~ 0.57 µM 0.26 µM 4.96 µM 6.60 µM
Ramos ~ 23.30 µM ~ 20.17 µM ~ 71.57 µM ~ 64.95 µM
PrestoBlue Cell
Viability Reagent
LIVE/DEAD Fixable
Aqua Dead Cell Stain
IC50 Drug Dose Response Table
Gambogic
Acid
Cell Type 19% Oxygen 1% Oxygen 19% Oxygen 1% Oxygen
Jurkat 0.60 µM 0.58 µM ~ 0.57 µM ~ 0.53 µM
Ramos 0.19 µM 0.19 µM ~ 0.30 µM ~ 0.33 µM
IC50 Drug Dose Response Table
LIVE/DEAD Fixable
Aqua Dead Cell Stain
PrestoBlue Cell
Viability Reagent
Hypoxia-Inducible Factor-1α (a mediator of oxygen
homeostasis) is expressed at higher levels in cells at 19%
oxygen, although only at higher concentrations.
B-cell lymphoma 2 (an anti-apoptotic protein involved in
the intrinsic pathway of apoptosis) expression is
dependent on drug compound.
Figure 6. Flow Analysis of HIF-1α expression Figure 7. Flow Analysis of Bcl-2 expression Figure 5b. Flow Analysis of Ramos cells treated with Amsacrine in 19% oxygen
Following treatment with Amsacrine, cells were incubated at either 19% Oxygen or 1% Oxygen in 5% CO2 at 37 ºC for 24 hrs. Cells were stained with HIF-1α,
CD95/FAS, and Bcl-2 antibodies, then analyzed on the Attune NxT. Three distinct populations are seen: live, dead, and unhealthy cells. Representative plots of
Bcl-2 % positive cells and HIF-1α mean fluorescence intensity are shown. Data is representative of triplicates in 2 separate experiments.
60 µ
M A
msa
cri
ne
0.0
6 µ
M A
msa
cri
ne
6 µ
M A
msa
cri
ne
Figure 5a. Flow Analysis of Jurkat cells treated with Amsacrine in 19% oxygen
60 µ
M A
msa
cri
ne
0.0
6 µ
M A
msa
cri
ne
6 µ
M A
msa
cri
ne
References 1. Lu, H.; Ouyang, W.; and Huang C. (2006). Inflammation, a Key Event in Cancer Development. Mol Cancer Res. 4(4): 221-233
2. Baig, S.; Seevasant, I.; Mohamad, J.; Mukheem, A.; Huri, H.Z.; and Kamarul, T. (2016). Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand? Cell Death and
Disease. 7, e2058
