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©2012, Promega Corporation.
Cell-Based Assays to Detect the Mechanism of Toxicity
March, 2013 [email protected]
©2012, Promega Corporation.
Cell Health Assays Using Plate Readers
CellTiter 96®
(MTT)
CellTiter 96®
AQueous (MTS)
CytoTox 96®
(LDH)
CellTiter 96®
AQueous One Soln
(MTS)
CytoTox-ONE™
(LDH)
CellTiter-Fluor™
CytoTox-Fluor™
(protease)
CytoTox-Glo™
(Protease)
MultiTox Fluor
MultiTox-Glo
Apo-ONE®
Caspase Assay
Caspase-Glo®
3/7
Caspase-Glo®
8 & 9
ApoLive-Glo™
CellTiter-Blue®
ADME
•P450-Glo™
•MAO-Glo™
•UGT-Glo™
•Pgp-Glo™
GSH-Glo™
GSH/GSSG-Glo™
ApoTox-Glo™
Mitochondrial ToxGlo™ Colorimetric Fluorescence Bioluminescence
Fluorescence & Bioluminescence
CellTiter-Glo®
Ultra-Glo™ Luciferase
CellTox™ Green
CellTiter-Glo®
One Solution
©2012, Promega Corporation.
Which Assay Should I Use? …First Decide What You Want to Measure
• Number of living cells (viability assay)
• Number of dead cells (cytotoxicity assay)
• How did the cells die
• Apoptosis vs. Necrosis
• Mitochondrial toxicity
• Oxidative stress
• Metabolic change
• Specific gene expression
• Multiplexing more than one parameter
©2012, Promega Corporation.
Cell Health Assays Overview
Viable cells detected using markers of active metabolism • Cellular conversion of indicator dyes (MTT / MTS / Resazurin)
• Protease marker
• ATP content
Dead cells detected using marker of membrane integrity • LDH release
• Protease release
• Dye uptake / staining
Apoptosis detected using caspase activities
Biochemical markers of cell stress leading to cytotoxicity • Mitochondrial toxicity
• Oxidative stress (ROS and GSH:GSSG ratio)
• NADH
Luciferase reporters of cell stress pathways leading to cytotoxicity
5
©2012, Promega Corporation.
Metabolic & Enzymatic Indicators of Cell Viability
Dead Cell Viable Cell
Reagent
Substrate Substrate No Rxn
X Product
Tetrazolium Reagents • MTT, MTS, XTT, WST Redox Indicators • Resazurin Enzyme Substrates • Protease Substrates
Active Metabolism or Protease
Incubation Step
Loss of Function
©2012, Promega Corporation.
ATP Assay for Cell Viability
ATP
Dead Cell
ADP
Viable Cell
Light No Reaction
Luciferin + Luciferase
CellTiter-Glo Reagent
X
• Lysis Solution • ATPase Inhibitors • Luciferin • UltraGlo Luciferase
ATP
©2012, Promega Corporation.
Advantages & Disadvantages of Viability Assays
Assay Advantages Disadvantages
MTT / MTS Widely used Inexpensive
MTT has 2 step protocol 1-4 hour incubation Interference by reducing compounds Toxic to cells Limited sensitivity
Resazurin Inexpensive Fluorescent readout Good sensitivity
1-4 hour incubation Interference by reducing compounds Toxic to cells Fluorescence interference
Protease 30 min protocol Cells remain viable Better sensitivity than resazurin Good choice for multiplexing
Fluorescence interference
ATP 10 min protocol Best sensitivity No fluorescence interference Lysis step stops reaction immediately (no incubation with viable cells)
Lytic protocol dictates sequence for multiplexing
8
©2012, Promega Corporation.
Future Improvements of CellTiter-Glo Formulation
“CellTiter-Glo 3D”
• Improved lytic capacity for 3D culture models
“CellTiter-Glo 2.0” (Abstract # TP157)
• Improved liquid reagent stability upon storage
“ViralTox-Glo”
• Validated for measuring viral cytopathic effect
9
22⁰C 4⁰C
CellTiter-Glo® 12 hours 3.5 days
new reagent 2 weeks 4 months*
10% change in performance
©2012, Promega Corporation.
Balb 3T3 Cells Treated with MTT for 4 Hours
10
Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences.
Same field of cells imaged immediately after addition of MTT and after 4 hours incubation.
©2012, Promega Corporation.
Balb 3T3 Cells Treated with GF-AFC for 4 Hours
13
Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences.
©2012, Promega Corporation.
Detecting Dead Cells: Two Basic Approaches
14
Viable Dead
The functional definition of cell viability is based on whether the
outer membrane is intact.
Dye Enzyme Marker
©2012, Promega Corporation.
Cell Health Assays Overview
Viable cells detected using markers of active metabolism • Cellular conversion of indicator dyes (MTT / MTS / Resazurin)
• Protease marker
• ATP content
Dead cells detected using marker of membrane integrity • LDH release
• Protease release
• Dye uptake / staining
Apoptosis detected using caspase activities
Biochemical markers of cell stress leading to cytotoxicity • Mitochondrial toxicity
• Oxidative stress (ROS and GSH:GSSG ratio)
• NADH
Luciferase reporters of cell stress pathways leading to cytotoxicity
15
©2012, Promega Corporation.
DNA Dye Staining to Detect Dead Cells (Overcomes some limitations of short half-life markers)
Dead Cell Viable Cell
Dye is excluded from live cells
DNA dye only stains nucleus of “dead” cells or debris
Non-permeable
DNA dye
18
Staining of dead
cells results in a
fluorescent signal
that is stable. X
©2012, Promega Corporation.
CellTox-Green Dye is Not Toxic to Cells
ATP assay data showing viability of cells exposed to bortezomib for 72 hrs • O cells exposed to DNA binding dye for 72 hr
• □ cells exposed to DNA binding dye for 15 min
19
©2012, Promega Corporation.
HeLa Cells Treated with CellTox-Green DNA Dye for 4 Hours
20
Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences.
©2012, Promega Corporation.
Reading the Same Plate Multiple Times to Detect the Onset of Cell Death
5000 K562 cells in 96 well plate
First appearance of cell death may trigger further experimentation with the same sample (e.g. How did the cells die? …apoptosis?)
©2012, Promega Corporation.
Samples with CellTox Green can be Multiplexed with Cell Viability and Apoptosis Assays
24hr
©2012, Promega Corporation.
Triplex at each time point Endpoint multiplexes possible at first emergence of toxicity
24hr 48hr
72hr
Note progression of cytotoxicity and gradual decline in caspase activity suggesting loss of enzymatic activity after release from dead cells.
©2012, Promega Corporation.
Advantages & disadvantages of assays to detect dead cells
Assay Advantages Disadvantages
LDH release
Widely used and accepted Absorbance or fluorescent options
Limited sensitivity Limited half-life of LDH in medium
Protease release
Designed for multiplexing More sensitive than LDH Fluorescent reagent is simpler than formulation for LDH assay Fluorescent or luminescent options
Limited half-life of protease marker Fluorescence interference (fluorescent format only)
DNA Staining
Non-toxic / real time assay Staining persists for 72 hours Good choice for multiplexing
Fluorescence interference Less sensitive than amplified protease release assay
25
CellTox™ Green Assay: Multiplexing a Fluorescent Assay with Luminescent Assays
CellTiter-Fluor™ Viability
Assay
CellTox Green™ Cytotoxicity
Assay
BacTiter-Glo™ Assay
CellTiter-Glo® Cell Viability
Assay
GSH-Glo™ & GSH/GSSG-Glo™
Assays
P450-Glo™ Cell-Based
Assays
Glo Reporter
Assays Will Work
Will work
Caspase-Glo® Assays
All possible with the GloMax®-Multi & -Multi+ Detection Systems
26
Probable Will work
NAD(P) / NAD(P)H-Early Access
Will work
CytoTox-Glo™ Assay
HDAC-Glo™ Assay
ROS Early Access
Glo Reporter Assay Multiplexes include: Nano-Glo™ One-Glo™ Bright-Glo™ Steady-Glo®
©2012, Promega Corporation.
Assays to Determine Cell Stress Events Leading to Toxicity
©2012, Promega Corporation.
Determining Mechanisms Leading to Cytotoxicity
Going beyond the standard assays available to detect live or dead cells.
Assay chemistries and approaches to detect…
• Apoptosis
• Mitochondrial toxicity
• Oxidative stress (ROS and GSH:GSSG ratio)
• Metabolic markers (NADH, NADPH)
• Gene expression in several stress-related pathways
28
©2012, Promega Corporation.
Detecting Apoptosis as the Mechanism of Cell Death
©2012, Promega Corporation.
Cell Health Assays Overview
Viable cells detected using markers of active metabolism • Cellular conversion of indicator dyes (MTT / MTS / Resazurin)
• Protease marker
• ATP content
Dead cells detected using marker of membrane integrity • LDH release
• Protease release
• Dye uptake / staining
Apoptosis detected using caspase activities
Biochemical markers of cell stress leading to cytotoxicity • Mitochondrial toxicity
• Oxidative stress (ROS and GSH:GSSG ratio)
• NADH
Luciferase reporters of cell stress pathways leading to cytotoxicity
30
©2012, Promega Corporation.
Common Apoptosis Assays
• Observing morphological features
• TUNEL
• Sub Go peak of DNA using flow cytometry
• Annexin V binding to exposed PS (flow cytometry)
• Caspase-3/7 activity
31
©2012, Promega Corporation.
O
C H 3
H N O Z - D E V D
O
O
O
H N N H D V E D - Z Z - D E V D
C a s p a s e 3
C a s p a s e 3
F l u o r e s c e n c e
F l u o r e s c e n c e
O
C H 3
H 2 N O
O
O H
O
H 2 N N H 2
C a s p a s e 3
S
N
N
S
H N C O O Z - D E V D
L u m i n e s c e n c e L u c i f e r a s e + A T P
S
N
N
S
H 2 N C O O
32
AMC, R110 and aminoluciferin substrates for measuring caspase activity
©2012, Promega Corporation.
Luminescent Caspase Assay
Dead Cell Viable Cell
Caspase-Glo® Reagent
Reagent No Rxn X
Reagent No Rxn X
Apoptotic Cell
Reagent Luminescence
• Lysis Solution
• Z-DEVD-aminoluciferin
• Stable Luciferase
• ATP
Pro-Caspase Inactive
Inactive Caspase
Active Caspase
©2012, Promega Corporation.
Caspase-Glo® 3/7 Time Course Indicates Caspase Activity is Transient
0 20 40 60 80 100
Ta m o x i fe n (µ M )
0
100
200
300
400
(Thousands)
Lum
ines
cenc
e
24 hr
6 hr
4 hr
2 hr
1 hr
0 hr
Caspase Act ivit y
Assay & Drug Devel Tech 2(1): 51, 2004
Cells are apoptotic
at 1 hr treatment
with 150µM Tamox
Caspase activity
decreases after 24
hours incubation
Tamoxifen Treatment of HepG2 Cells
©2012, Promega Corporation.
Luminescent Caspase-3/7 Assay
Advantages:
• Homogeneous (add-mix-measure)
• 50-100X greater sensitivity than fluorescent assays
• No interference by fluorescent compounds
• Flexible incubation time to record “glow” signal
Disadvantages:
• Average of entire well (not individual cells)
• Caspase is a transient marker
• Possibility of inhibition of luciferase by test compounds; but it is probably less of a problem than using a fluorescent assay
©2012, Promega Corporation.
Detecting Mitochondrial Toxicity
• ATP can be used as a marker of functioning mitochondria
• Net ATP production from glycolysis can be blocked by using glucose-free medium*
• Decrease in ATP marker (without general necrosis) during 1-4hr incubation suggests mitochondrial toxicity
• ATP and membrane integrity assays can be multiplexed
36
X * Lisa D. Marroquin, James Hynes, James A. Dykens, Joseph D. Jamieson, and Yvonne Will. Circumventing the crabtree effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97:539 – 547, 2007.
©2012, Promega Corporation.
Mitochondrial toxicity can be detected by using controlled culture conditions and short incubation
• Cells are exposed to treatment less than 4hr to avoid necrosis from non-mitochondrial pathways
• Sequential multiplex protocol is used to detect
• Cell death (leakage of protease marker into medium indicating loss of membrane integrity)
• Mitochondrial function (ATP content)
• Decrease in ATP without change in cell viability suggests mitochondrial toxicity
38
©2012, Promega Corporation.
Mitochondrial ToxGloTM Assay Multiplex membrane integrity and ATP content
bis-AAF-R110
Substrate
ATP Assay
Reagent
Incubate 30 min
Incubate
Record Luminescence
Incubate 10 min
Record Fluorescence
Treat cells 30 min - 4hr
No toxicity at this exposure period
Primary Necrosis
Expected Assay Profiles
MitoTox Without necrosis
MitoTox with necrosis
Change to glucose-free medium + galactose
©2012, Promega Corporation.
Oxidative Stress Assays
©2012, Promega Corporation.
Oxidative Stress Assays
Oxidative stress: an imbalance between the production of reactive oxygen species (ROS) and the cell's capacity to detoxify the ROS or to repair the oxidative damage.
Markers of oxidative stress: • Altered GSH:GSSG ratio (lowered GSH, increased GSSG) • ROS (super oxide, hydroxyl radical, nitric oxide, hypochlorite convert
to more stable H2O2)
41
©2012, Promega Corporation.
GSH Assay as Marker for Oxidative Stress
• Reduced form of glutathione (GSH) serves as an antioxidant in cells
• Decreased levels of GSH are associated with oxidative stress
• GSH and GSSG can be measured separately with a luminescent assay using Glutathione S Transferase (GST) and luciferase
• A fluorescent cell viability assay can be sequentially multiplexed with the luminescent GSH assay
42
©2012, Promega Corporation.
Principal of GSH:GSSG Ratio Assay (Assays must be run in parallel in separate wells.)
43
Total Glutathione
GSSG
GSH
Reduce With DTT
Oxidized GSSG
GSH
Block with NEM
GSSG GSH Reduce
43
HO S
N
S
NCOOH
CH3
S
O
OO
NO2
S
N
S
NCOOH
GST
GS-R
GSH
Luciferase, ATP (LDR)
Light
©2012, Promega Corporation.
Menadione Treatment Drops GSH:GSSG Ratio
GSH:GSSG changes indicate: • Oxidative Stress • Compound toxicity • Reactive metabolite formation
©2012, Promega Corporation.
Reactive Oxygen Species (ROS) Assay
©2012, Promega Corporation.
ROS-Glo H2O2 Assay (coming in 2013)
• Direct H2O2 detection without using Horseradish Peroxidase (HRP) • Mitigates HRP mediated false hits
• Homogeneous Bioluminescent Assay
• Add-mix-read • No fluorescence interference
• Cell based assay
• Detects H2O2 content of culture wells
©2012, Promega Corporation.
ROS-Glo Assay Chemistry Based on Pro-Luciferin
H2O2
LDR
• Self-cleaving linker
• D-Cys cyclization
Luciferase
Modified Pro-luciferin
Light
©2012, Promega Corporation.
ROS-Glo™ H2O2 Assay Protocol
49
Incubate up to 2 hours
Add test compound
and modified pro-luciferin
peroxide detector
Add luciferin
detection reagent
Incubate 15 min
Record luminescence
ROS-Glo™ H2O2 Assay of Hep G2 Cells Treated with Menadione
©2012, Promega Corporation.
Luminescent ROS Assay
• Commercial product is still in development
• Optional cell-based or enzymatic assay format
• Can sample culture medium and multiplex a cell viability assay
• Culture medium can affect level of ROS production
• Comparison of Amplex Red (requiring horseradish peroxidase) and ROS-Glo for screening LOPAC demonstrated fewer false positives with luminescent assay
51
©2012, Promega Corporation.
Assays for NADH/NADPH, NAD+, & NADP Metabolic Indicators
©2012, Promega Corporation.
Bioluminescent Assays for Adenine Dinucleotides
Bioluminescent assays are being developed for:
1. NADH + NADPH (total of both reduced forms)
2. NAD+ + NADH (total non-phosphorylated)
3. NADP + NADPH (total non-phosphorylated)
Additional information on Poster # TP159
54
©2012, Promega Corporation.
Basic Principle for the Bioluminescent Adenine Dinucleotide Detection Assays
Diaphorase
NAD(P)H NA(D)P+
Modified Luciferin
Luciferin
Proluciferin substrate couples NADH or NADPH to production of luciferin used to generate light
Assay chemistry does not discriminate between NADH and NADPH Detects only the reduced forms
©2012, Promega Corporation.
Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides
56
Lactate dehydrogenase and lactate are used to measure total non-phosphorylated NAD+ + NADH
Glucose-6-phosphate and G-6-PDH are used to measure total phosphorylated NADP + NADPH
Diaphorase
NADPH NADP+
Proluciferin Luciferin
G-6-P Product
G-6-PDH
Diaphorase
NADH NAD+
Proluciferin Luciferin
Lactate Product
LDH
©2012, Promega Corporation.
Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides
57
Lactate dehydrogenase and lactate are used to measure total non-phosphorylated NAD+ + NADH
Glucose-6-phosphate and G-6-PDH are used to measure total phosphorylated NADP + NADPH
©2012, Promega Corporation.
Bioluminescent Assay Approach is More Sensitive
58
©2012, Promega Corporation.
Summary of NAD(P) / NAD(P)H Assays
59
Bioluminescence assay to measure NAD(P) and NAD(P)H based on the reduction of proluciferin by diaphorase
One-step homogeneous 30 min cell-based assay In general, ~50X more sensitive than fluorescent assay Wide linear range; large signal window; good S/B Can measure upstream events in cancer cell metabolism that are coupled to
NAD(P)/NAD(P)H production
Assay NADH /NADPH NAD/NADH NADP/NADPH
Application
Enzyme activity assays; Direct
detection of NADH & NADPH in cells
Total NAD + NADH using homogenous one step assay; NAD:NADH ratio
after acid/base separation
Total NADP + NADPH using homogenous one step assay;
NADP:NADPH ratio after acid/base separation
©2012, Promega Corporation.
Cell Stress Response Pathway Reporters
©2012, Promega Corporation.
Stress Response Pathways Leading to Cytotoxicity
• Stress response pathways are toxin activated signal transduction events that modulate transcription factors to trigger expression of cytoprotective genes to enable the cell to attempt to restore homeostasis.*
• Triggering cell response pathways occurs at lower toxin doses or exposure times than what is needed to trigger necrosis or apoptosis.
• If stress cannot be overcome to re-establish homeostasis, the result is induction of apoptosis and removal of the cell.
*Simmons, S.O. et al., Cellular stress response pathway system as a sentinel ensemble in toxicological screening. Tox. Sci. 111(2): 202-225, 2009.
61
©2012, Promega Corporation.
Example Stress Response Pathways
Oxidative Stress Response: Signaling pathway that leads to Nrf2 transcription factor binding to antioxidant response elements (ARE) that induce expression of genes to neutralize Reactive Oxygen Species (ROS) and limit oxidative damage to cellular components. (Among most commonly studies pathways)
Heat Shock Response: HSF-1 activates expression of Hsp70 & Hsp27 chaperones that bind to and facilitate refolding of denatured proteins.
62
©2012, Promega Corporation.
Skin Sensitization Model Pathway KeratinoSens Cell Line from Givaudan
63
Modified from Fig. 2 from Natsch, A. Tox. Sci. 113(2), 284–292 (2010)
©2012, Promega Corporation.
Stress and Toxicity Pathway Vectors
64
Pathway/Response Transcription Factor Name
Antioxidant Nrf2 pGL4/ARE DNA damage p53 pGL4/p53
ER stress ATF6 pGL4/ERSE ER Stress ATF4 pGL4/ATF4 ER stress Xbp1 pGL4/Xbp1
Heavy metal stress MTF1 pGL4/MRE
Heat shock HSF1 pGL4/HSE Hypoxia Hif1α pGL4/HRE p38/JNK AP1 pGL4/AP1
Xenobiotic stress AhR pGL4/XRE
Inflamation NFκB Cat # E8491 Osmotic stress NFAT5 pGL4/NFAT5
All constructs with pGL4.27 backbone [luc2P/minP/HygR]
©2012, Promega Corporation.
Cell lines available as custom products http://www.promega.com/a/forms/custom-assays/custom-assay-services.html
65