Poster: New and improved cellular health evaluation of 2D...
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Thermo Fisher Scientific • 29851 Willow Creek Road• Eugene, OR 97402 • thermofisher.com For Research Use Only 0.1 1 10 100 0 2 4 6 8 0 500 1000 1500 2000 Niclosamide (uM) Varioskan LUX Fluorescence 500/530 nm High Content CX7 Fluorescence 500/530 nm 1 10 100 1000 0.0 0.5 1.0 1.5 0 1000 2000 3000 4000 Gambogic Acid (uM) Varioskan LUX Fluorescence 500/530 nm High Content CX7 Fluorescence 500/530 nm 1 10 100 1000 0 1 2 3 4 5 6 Spheroid GA treatment 26 hours [ 1X ] CyQUANT XTT Gambogic Acid (uM) Absorbance XTT (450-660 nm) 1 10 100 1000 0 2 4 6 Spheroid GA treatment 26 hours [ 2X ] CyQUANT XTT Gambogic Acid (uM) Absorbance XTT (450-660 nm) RESULTS Spheroids in Cancer Drug Discovery Relevant pharmacology & better tumor models ABSTRACT High throughput screening (HTS) is an effective method for identifying putative active compounds for therapeutics. Assays that evaluate changes in cellular functions are essential for characterizing test compounds and their potential role as pharmaceuticals. Researchers in drug discovery and cancer research rely on reproducible biological assays to guide medicinal chemistry programs. A current goal in the cancer research field is to explore and expand the use of microplate readers and assay systems into the realm of 3D cellular models for drug discovery in anticancer research, specifically aimed at modelling solid tumors to screen compounds and confirm their activity. In this study, we screened multiple therapeutic drugs using different HTS assays to establish drug dose response curves and to understand the diversity in cell health assays. Despite the lack of guidelines and enabling technologies to study 3D cellular models, we establish the different impact therapeutics have on 2D versus 3D cellular models using existing cellular assays. INTRODUCTION The ability to use diverse cell health assays allows researchers to monitor different cell health read-outs and target specific cellular responses. The differences in potency and drug incubation time between 2D and 3D models indicate that drug effectiveness is dependent on cellular microenvironment. The ability to use existing microplate tools that are established for 2D cell models in 3D cell models provides a time and cost efficient basis for understanding therapeutic effects on 3D cellular models, allowing for rapid therapeutic characterization in drug discovery with minimal investment. MATERIALS AND METHODS When comparing existing cell health assays, we evaluated the differences between PrestoBlue TM HS and PrestoBlue. The PrestoBlue HS assay has a larger dynamic range, minimal background signal, and allows the removal of hotspots (false-positives). The PrestoBlue HS assay has the lowest detection threshold among the resazurin-based fluorescent assays available for detection of cell viability. With this advantage, the limit of quantification can be studied. Additionally, as an improved reduction potential assay, PrestoBlue HS can be used to interrogate the differences between 2D and 3D cellular models. We demonstrate that existing microplate assays, such as PrestoBlue High Sensitivity (HS), CyQUANT® XTT, and CyQUANT® Direct, can be used to quantify functional differences between 2D and 3D cellular models. Using all three cell health assays, we found that a longer incubation time with drug is required to have an effect on the health of 3D models compared to 2D models. These results suggest that drug treatment is less potent in 3D models than 2D models, as demonstrated by a shift in the apparent IC50 values. Additionally, the dense compact structure of 3D models is preserved at low to no concentrations of drug, indicating that higher concentrations of drug are required to kill cells grown in 3D culture compared to 2D monolayers. Leticia A. Montoya, Bhaskar S. Mandavilli, and Madison Javitz, Thermo Fisher Scientific, 29851 Willow Creek Road, Eugene, Oregon, USA, 97405 New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays Working in 3D involves formation of spheroids. Spheroids are aggregates that can either be grown in suspension, encapsulated, or grown on the top of a 3D matrix. CellEvent TM Caspase 3/7 detection reagent measures cleaved caspase 3, hall mark of apoptosis cells The spheroid size measurements is a phenotypic readout to measure drug effects. Drug Potency on Monolayer (2D) and Spheroid (3D) Cellular Models: (A) As incubation time increases with gambogic Acid, the drug becomes more potent to 2D monolayer cells. Gambogic Acid is less potent to 3D spheroids than to 2D monolayer cells; shift in IC50 values. (B) High concentrations of Gambogic Acid drug are required to have an effect on spheroid health. Long incubation time (greater than 24 hours) is required with Gambogic Acid to effect Spheroid health. 2-3 hours 19 hours Treatment time with GA Optimization of viability reagents for 3D spheroid analysis 2 hours GA 24 hours GA 48 hours 72 hours Treatment time with GA Measuring Cellular Health on Monolayer (2D) and Spheroid (3D) Cellular Models First Step is determining concentration of reagent. CyQUANT XTT gives a better signal/noise ratio and same IC50 value when used at twice the concentration on Spheroids when compared to one times the concentration on monolayer cellular models. Second step is determining incubation time with reagent. The incubation time with reagents is extended when working with spheroids when compared to monolayer. Highly treated spheroids presents a very low signal turn-on, further confirming the presence of dead cells. Measuring Cellular Health on microplate reader Varioskan TM LUX versus High Content Analysis System CX7 2-3 hours 19 hours Treatment time with GA 1 10 100 1000 0 5 10 15 20 0 5000 10000 15000 20000 Gambogic Acid (uM) Varioskan LUX Flourescence 500/530 High Content CX7 Flourescence 500/530 LUX CX7 LUX IC 50 16.75 CX7 IC 50 19.76 Enhanced Assay Performance High Sensitivity Resazurin-based Reagents: An innovative purification process was developed that removes contaminants from the original PrestoBlue and alamarBlue products to make the High Sensitivity versions (HS) of both reagents. 1 10 100 1000 0 100 200 300 400 500 Gambogic Acid ( μM) Fluorescence 560/590 nm PrestoBlue HS PrestoBlue alamarBlue HS alamarBlue Resazurin-based Reagents 0 50 100 150 200 250 Signal/Background 10 60 120 Time (minutes) PrestoBlue (Thermo Fisher Scientific) PrestoBlue HS (Thermo Fisher Scientific) CellTiter-Blue® (Promega) Performance Comparison of resazurin-based reagents: (A) High Sensitivity reagents have a 100% increase in the signal-to-background ratio and greater dynamic range when compared to other resazurin-based reagents. (B) Cellular reduction kinetics on A549 spheroids (C) A549 spheroids were treated with Gambogic Acid for 24 hours and cell viability was measured with resazurin-based reagents (fluorescence response was measured at 60 minute). 0 2 4 6 8 0 500 1000 1500 2000 3D spheroids-on cells-viability-time course Time (hours) Fluorescence 560/590 nm PrestoBlue HS PrestoBlue alamarBlue HS alamarBlue CyQUANT XTT incubation time IC50s (μM) Concentration (μM) 1X 2X 4 hours 38.4 38.9 22 hours 36.5 35.5 51 hours 27.5 27.3 4 hours 51 hours 22 hours 4 hours 51 hours 22 hours 0.1 1 10 100 0 5000 10000 15000 0 500 1000 1500 2000 Niclosamide A549 cells Nicolsamide ( M) Cell Event Green (503/530 nm) MitoTracker Orange Cell Event Green MitoTracker Orange High-Content Screening Platforms provide special and temporal resolution. High-Content Screening Platforms are equipped with wide field or confocal optics to allow for ease of multiplexing. Assays for microplate readers allow for rapid quantification of cell health measurements and enzyme activity. 3D High Content Screening Platform: CellInsight TM CX7 untreated 6 μM 60 μM Niclosamide treated 3D spheroids 2D Monolayer 3D spheroids Cellular Activity Cell to Cell Cell to ECM Cellular Interactions Cellular Adhesion, Proliferation, & modified genes Proliferative Ring & Apoptotic core Micro- environment Immune Response therapy Gradients to Oxygen, metabolites & nutrients MitoTracker TM detection reagent measures mitochondria health in live cells. The reagent is dependent upon membrane integrity. Mitochondria health is inversely proportional to the apoptotic pathway. Assessing Spheroid Health using High Content Analysis untreated 6 μM 60 μM Gambogic Acid treated 3D spheroids Using microplater reader Varioskan TM LUX to drug screen before image analysis on HCA System Existing solution- and fluorescence-based assays can be analyzed by microplate readers for initial drug discovery questions. Similar IC50 values are determined by using existing assays on the microplate reader and High Content System. CONCLUSIONS Monolayer and Spheroids can be monitored across multiple platforms with multiple different reagents. Existing microplate assays can be used to quantify functional differences between 2D and 3D cellular models. Pharmaceutical drugs can be analyzed on a microplate as initial studies before further analysis on imaging platform using existing cell functional assays. Increase in Cell Viability CyQUANT Direct High Throughput Drug Screening. The IC50 values of multiple drugs were determined by multiplexing with CellEvent Green and MitoTracker Orange and analyzing on the Varioskan TM LUX microplate reader. CyQUANT TM Direct Green CellEvent TM Green LUX CX7 LUX CX7 MitoTracker TM Orange 0.01 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.15 0.20 0.25 0.30 0.35 Drug Treated Spheroids Cell Event Green Analysis Treated with drug 19 hours after plating Drug (uM) VarioskanLUX Fluorescence 500/530 nm VarioskanLUX Fluorescence 500/530 nm 0.01 0.1 1 10 100 1000 4 5 6 7 8 0 2 4 6 8 Drug Treated Spheroids MitoTracker Orange Treated with drug 19 hours after plating Drug (uM) VarioskanLUX Fluorescence 500/530 nm VarioskanLUX Fluorescence 500/530 nm Niclosamide Gambogic Acid Amide Gambogic Acid Nocodazole 1 10 100 1000 0 2 4 6 0 2 4 6 8 10 Oxydative Stress & DNA content Gambogic Acid (uM) CellROX Deep Red (644/665 nm) Hoechst (361/497 nm) 0 1 2 3 4 5 6 0 5 10 15 20 0 1 2 3 4 5 Fluorescence Based Assays 5k A549 spheroids Time (hours) CyQUANT Direct Green (500/530 nm) MitoTracker Orange (554/576 nm) CyQUANT Direct Green + 250 μM GA CyQUANT Direct Green (Untreated) MitoTracker Orange + 250 μM GA MitoTracker Orange (Untreated) 0 10 20 30 40 50 60 0 500 1000 1500 2000 0 2 4 6 Solution Based Cell Viability Assays 5k A549 spheroids Time (hours) PrestoBlue Fluorescence 560/590 nm CyQUANT XTT Absorbance 450-660 nm PrestoBlue (Untreated) PrestoBlue + 250 μM GA CyQUANT XTT (Untreated) CyQUANT XTT+ 250 μM GA CyQUANT TM Direct Green detection reagent measures DNA content and cytotoxicity of individual cells. Solution-based cell viability assays provide information on entire cell populations rather than tracking the behavior of individual cells. After some initial optimization, both types of reagents (solution and individual cell analysis) can be used to analyze cellular function and cellular viability on a microplate reader. CellInsight TM CX7 Varioskan TM LUX LUX IC 50 4.03 CX7 IC 50 1.53 Multiple Modes of cellular function can be monitored by microplate reader and existing cell functional assays CellROX Hoescht 1X = monolayer working concentration 2X = two times monolayer working concentration (A) (B) (C) (A) (B) (C) 1324-E LUX IC 50 22.08 CX7 IC 50 18.57 Varioskan LUX analysis © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified
Transcript of Poster: New and improved cellular health evaluation of 2D...
Thermo Fisher Scientific • 29851 Willow Creek Road• Eugene, OR 97402 • thermofisher.comFor Research Use Only
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RESULTSSpheroids in Cancer Drug DiscoveryRelevant pharmacology & better tumor models
ABSTRACT
High throughput screening (HTS) is an effective method for
identifying putative active compounds for therapeutics. Assays that
evaluate changes in cellular functions are essential for
characterizing test compounds and their potential role as
pharmaceuticals.
Researchers in drug discovery and cancer research rely on
reproducible biological assays to guide medicinal chemistry
programs. A current goal in the cancer research field is to explore
and expand the use of microplate readers and assay systems into
the realm of 3D cellular models for drug discovery in anticancer
research, specifically aimed at modelling solid tumors to screen
compounds and confirm their activity. In this study, we screened
multiple therapeutic drugs using different HTS assays to establish
drug dose response curves and to understand the diversity in cell
health assays. Despite the lack of guidelines and enabling
technologies to study 3D cellular models, we establish the different
impact therapeutics have on 2D versus 3D cellular models using
existing cellular assays.
INTRODUCTION
The ability to use diverse cell health assays allows
researchers to monitor different cell health read-outs and
target specific cellular responses. The differences in
potency and drug incubation time between 2D and 3D
models indicate that drug effectiveness is dependent on
cellular microenvironment. The ability to use existing
microplate tools that are established for 2D cell models in
3D cell models provides a time and cost efficient basis for
understanding therapeutic effects on 3D cellular models,
allowing for rapid therapeutic characterization in drug
discovery with minimal investment.
MATERIALS AND METHODS
When comparing existing cell health assays, we evaluated
the differences between PrestoBlueTM HS and PrestoBlue.
The PrestoBlue HS assay has a larger dynamic range,
minimal background signal, and allows the removal of
hotspots (false-positives). The PrestoBlue HS assay has
the lowest detection threshold among the resazurin-based
fluorescent assays available for detection of cell viability.
With this advantage, the limit of quantification can be
studied. Additionally, as an improved reduction potential
assay, PrestoBlue HS can be used to interrogate the
differences between 2D and 3D cellular models.
We demonstrate that existing microplate assays, such as
PrestoBlue High Sensitivity (HS), CyQUANT® XTT, and
CyQUANT® Direct, can be used to quantify functional
differences between 2D and 3D cellular models. Using all
three cell health assays, we found that a longer incubation
time with drug is required to have an effect on the health of
3D models compared to 2D models. These results suggest
that drug treatment is less potent in 3D models than 2D
models, as demonstrated by a shift in the apparent IC50
values. Additionally, the dense compact structure of 3D
models is preserved at low to no concentrations of drug,
indicating that higher concentrations of drug are required to
kill cells grown in 3D culture compared to 2D monolayers.
Leticia A. Montoya, Bhaskar S. Mandavilli, and Madison Javitz,
Thermo Fisher Scientific, 29851 Willow Creek Road, Eugene, Oregon, USA, 97405
New and improved cellular health evaluation of 2D and 3D cellular models
using microplate reader assays
Working in 3D involves
formation of spheroids.
Spheroids are aggregates
that can either be grown in
suspension, encapsulated,
or grown on the top of a
3D matrix. CellEventTM Caspase 3/7 detection reagent measures cleaved caspase 3, hall mark of apoptosis cells
The spheroid size measurements is a phenotypic readout to measure drug effects.
Drug Potency on Monolayer (2D) and Spheroid (3D) Cellular Models: (A) As incubation time increases with
gambogic Acid, the drug becomes more potent to 2D monolayer cells. Gambogic Acid is less potent to 3D spheroids
than to 2D monolayer cells; shift in IC50 values. (B) High concentrations of Gambogic Acid drug are required to have
an effect on spheroid health. Long incubation time (greater than 24 hours) is required with Gambogic Acid to effect
Spheroid health.
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M o n o la y e rT r e a t e d w i t h 1 0 u L P B fo r 2 1 h r s
T r e a t m e n t w i th d r u g 1 9 h r s a f te r p la t in g
G a m b o g ic A c id (u M )
RF
U (
56
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+/
- S
D
2 -3 h o u rs
1 9 h o u rs
EC50
2-3 hours
8.588
19 hours
3.359
in c u b a t io n t im e w i t h G ATreatment time with GA
Optimization of viability reagents for 3D spheroid analysis
1 1 0 1 0 0 1 0 0 0
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3 0 0
S p h e r o idT r e a t e d w i t h 1 0 u L PB
T r e a t e d w i th d r u g 1 9 h o u r s a f te r p la t in g
G a m b o g ic A c id (u M )
Flu
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nm 2 h o u rs G A
2 4 h o u rs G A
4 8 h o u rs
7 2 h o u rs
EC50
2 hours GA
156.2
24 hours GA
53.37
48 hours
28.44
72 hours
25.95
Treatment time with GA
Measuring Cellular Health on Monolayer (2D) and Spheroid (3D) Cellular Models
First Step is determining concentration of reagent. CyQUANT XTT gives a better signal/noise ratio and same
IC50 value when used at twice the concentration on Spheroids when compared to one times the concentration on
monolayer cellular models.
Second step is determining incubation time with reagent. The incubation time with reagents is extended
when working with spheroids when compared to monolayer. Highly treated spheroids presents a very low
signal turn-on, further confirming the presence of dead cells.
Measuring Cellular Health on microplate reader VarioskanTM LUX versus High Content Analysis System CX7
0 .1 1 1 0 1 0 0
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T r e a t m e n t w i th d r u g 1 9 h r s a f te r p la t in g
G a m b o g ic A c id (u M )
RF
U (
56
0/
59
0)
+/
- S
D
2 -3 h o u rs
1 9 h o u rs
EC50
2-3 hours
8.588
19 hours
3.359
in c u b a t io n t im e w i t h G ATreatment time with GA
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T r e a tm e n t w i t h d r u g f o r 4 8 h r s
G a m b o g ic A c id (u M )
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C X 7
IC50
LUX
~ 16.75
CX7
19.76
LUX
IC50 16.75
CX7
IC50 19.76
Enhanced Assay Performance
High Sensitivity Resazurin-based Reagents: An innovative purification process was developed that removes
contaminants from the original PrestoBlue and alamarBlue products to make the High Sensitivity versions (HS)
of both reagents.
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Flu
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P re s to B lu e H S
P re s to B lu e
a la m a rB lu e H S
a la m a rB lu e
IC50
PrestoBlue HS
17.64
PrestoBlue
21.86
alamarBlue HS
20.00
alamarBlue
17.05
1 1 0 1 0 0 1 0 0 0
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T ra n s fo rm o f 3 D s p h e ro id s -o n c e lls -P B H S , P B , a B H S , a B -d ru g d o s e -6 0 m in in c u b a t
G a m b o g ic A c id (µ M )
Flu
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0/5
90
nm
P re s to B lu e H S
P re s to B lu e
a la m a rB lu e H S
a la m a rB lu e
IC50
PrestoBlue HS
17.64
PrestoBlue
21.86
alamarBlue HS
20.00
alamarBlue
17.05
Resazurin-based Reagents
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inu
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ou
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2 h
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C e llT ite r -B lu e
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P re s to B lu e 1 .0
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PrestoBlue (Thermo Fisher Scientific)
PrestoBlue HS (Thermo Fisher Scientific)
CellTiter-Blue® (Promega)
Performance Comparison of resazurin-based reagents: (A) High Sensitivity reagents have a 100% increase
in the signal-to-background ratio and greater dynamic range when compared to other resazurin-based
reagents. (B) Cellular reduction kinetics on A549 spheroids (C) A549 spheroids were treated with Gambogic
Acid for 24 hours and cell viability was measured with resazurin-based reagents (fluorescence response was
measured at 60 minute).
0 2 4 6 8
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21.86
alamarBlue HS
20.00
alamarBlue
17.05
CyQUANT XTT
incubation time
IC50s
(µM)
Concentration
(µM)
1X 2X
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22 hours 36.5 35.5
51 hours 27.5 27.3
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[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
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an
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T (
45
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)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
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5 1 h r
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an
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XT
T (
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0-6
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nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
4 hours
51 hours
22 hours
1 1 0 1 0 0 1 0 0 0
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S p h e ro id
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[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
1 1 0 1 0 0 1 0 0 0
0
1
2
3
4
5
6
S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
4 hours
51 hours
22 hours
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C e ll E v e n t G re e n
M ito T ra c k e r O ra n g e
High-Content Screening Platforms provide special and temporal
resolution.
High-Content Screening Platforms are equipped with wide field or
confocal optics to allow for ease of multiplexing.
Assays for microplate readers allow for rapid quantification of cell
health measurements and enzyme activity.
3D High Content Screening Platform: CellInsightTM CX7
untreated6 µM60 µM
Niclosamide treated 3D spheroids2D Monolayer 3D spheroids
Cellular
Activity
Cell to Cell Cell to ECM
Cellular
Interactions
Cellular Adhesion,
Proliferation, & modified genes
Proliferative Ring &
Apoptotic core
Micro-
environment
Immune Response therapy Gradients to Oxygen,
metabolites &
nutrients
MitoTrackerTM detection reagent measures
mitochondria health in live cells. The reagent is
dependent upon membrane integrity.
Mitochondria health is inversely proportional to
the apoptotic pathway.
Assessing Spheroid Health using High Content Analysis
untreated6 µM60 µM
Gambogic Acid treated 3D spheroids
Using microplater reader VarioskanTM LUX to drug screen before image analysis on HCA System
Existing solution- and fluorescence-based assays can be analyzed by microplate readers for initial drug
discovery questions. Similar IC50 values are determined by using existing assays on the microplate reader and
High Content System.
CONCLUSIONS
Monolayer and Spheroids can be monitored across multiple platforms with multiple different
reagents.
Existing microplate assays can be used to quantify functional differences between 2D and 3D
cellular models.
Pharmaceutical drugs can be analyzed on a microplate as initial studies before further
analysis on imaging platform
using existing cell functional
assays.
Increase in Cell Viability
CyQUANT Direct
High Throughput Drug Screening. The IC50 values of multiple
drugs were determined by multiplexing with CellEvent Green and
MitoTracker Orange and analyzing on the VarioskanTM LUX
microplate reader.
CyQUANTTM Direct Green CellEventTM Green
1 1 0 1 0 0 1 0 0 0
0
5
1 0
1 5
2 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
S p h e r o idT r e a t e d w i t h 1 x C Q D fo r 3 h r s
T r e a tm e n t w i t h d r u g f o r 4 8 h r s
G a m b o g ic A c id (u M )
Va
rio
sk
an
LU
X
Flo
ure
sc
en
ce
50
0/5
30
Hig
h C
on
ten
t CX
7
Flo
ure
sc
en
ce
50
0/5
30
L U X
C X 7
IC50
LUX
~ 16.75
CX7
19.76
1 1 0 1 0 0 1 0 0 0
0
5
1 0
1 5
2 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
S p h e r o idT r e a t e d w i t h 1 x C Q D fo r 3 h r s
T r e a tm e n t w i t h d r u g f o r 4 8 h r s
G a m b o g ic A c id (u M )
Va
rio
sk
an
LU
X
Flo
ure
sc
en
ce
50
0/5
30
Hig
h C
on
ten
t CX
7
Flo
ure
sc
en
ce
50
0/5
30
L U X
C X 7
IC50
LUX
~ 16.75
CX7
19.76
MitoTrackerTM Orange
0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0
0 .0
0 .2
0 .4
0 .6
0 .1 5
0 .2 0
0 .2 5
0 .3 0
0 .3 5
D r u g T r e a te d S p h e r o id s
C e ll E v e n t G re e n A n a ly s isT r e a t e d w i th d r u g 1 9 h o u r s a f te r p la t in g
D ru g (u M )
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
N ic lo s a m id e (u M )
G a m b o g ic A c id A m id e (u M )
G a m b o g ic A c id (u M )
N o c o d a z o le (u m )
0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0
4
5
6
7
8
0
2
4
6
8
D r u g T r e a te d S p h e r o id s
M ito T r a c k e r O r a n g eT r e a te d w i th d r u g 1 9 h o u r s a fte r p la t in g
D ru g (u M )
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
N ic lo s a m id e (u M )
G a m b o g ic A c id A m id e (u M )
G a m b o g ic A c id (u M )
N o c o d a z o le (u m )
0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0
4
5
6
7
8
0
2
4
6
8
D r u g T r e a te d S p h e r o id s
M ito T r a c k e r O r a n g eT r e a te d w i th d r u g 1 9 h o u r s a fte r p la t in g
D ru g (u M )
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
N ic lo s a m id e
G a m b o g ic A c id A m id e
G a m b o g ic A c id
N o c o d a z o le
1 1 0 1 0 0 1 0 0 0
0
2
4
6
0
2
4
6
8
1 0
O x y d a t iv e S tre s s & D N A c o n te n t
G a m b o g ic A c id (u M )
Ce
llR
OX
De
ep
Re
d (
64
4/6
65
nm
)
Ho
ec
hs
t (36
1/4
97
nm
)
0 1 2 3 4 5 6
0
5
1 0
1 5
2 0
0
1
2
3
4
5
F lu o r e s c e n c e B a s e d A s s a y s
5 k A 5 4 9 s p h e r o id s
T im e (h o u rs )
Cy
QU
AN
T D
ire
ct
Gre
en
(50
0/5
30
nm
)
Mit
oT
ra
ck
er O
ra
ng
e
(55
4/5
76
nm
)
2 6 0 G A u M -D ire c t
2 5 0 u M M ito O R
0 u M -M ito O R
0 G A u M -D ire c t
2 5 0 u M C e llR o x
0 u M C e llR O X
CyQUANT Direct Green + 250 µM GA
CyQUANT Direct Green (Untreated)
MitoTracker Orange + 250 µM GA
MitoTracker Orange (Untreated)
0 1 0 2 0 3 0 4 0 5 0 6 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
0
2
4
6
S o lu t io n B a s e d C e ll V ia b ility A s s a y s5 k A 5 4 9 s p h e r o id s
T im e (h o u rs )
Pre
sto
Blu
e
Flu
ore
sc
en
ce
56
0/5
90
nm
Cy
QU
AN
T X
TT
Ab
so
rb
an
ce
45
0-6
60
nm
PrestoBlue (Untreated)
PrestoBlue + 250 µM GA
CyQUANT XTT (Untreated)
CyQUANT XTT+ 250 µM GA
CyQUANTTM Direct Green detection reagent measures DNA content and
cytotoxicity of individual cells.
Solution-based cell viability assays provide information on entire cell
populations rather than tracking the behavior of individual cells.
After some initial optimization, both types of reagents (solution and individual
cell analysis) can be used to analyze cellular function and cellular viability on
a microplate reader.
CellInsightTM CX7VarioskanTM LUX
LUX
IC50 4.03
CX7
IC50 1.53
Multiple Modes of cellular function can
be monitored by microplate reader and
existing cell functional assays
CellROX
Hoescht
1X = monolayer working concentration
2X = two times monolayer working concentration
(A) (B) (C)
(A)(B) (C)
1324-E
LUX
IC50 22.08
CX7
IC50 18.57
Varioskan LUX analysis
© 2019 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher
Scientific and its subsidiaries unless otherwise specified
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