cell-based assays to detect the mechanism of toxicity/media/files/promega worldwide/europe/promega...

Cell-Based Assays to Detect the Mechanism of Toxicity

March, 2013 Terry.riss@Promega.com

Cell Health Assays Using Plate Readers

CellTiter 96®

AQueous (MTS)

CytoTox 96®

CellTiter 96®

AQueous One Soln

CytoTox-ONE™

CellTiter-Fluor™

CytoTox-Fluor™

(protease)

CytoTox-Glo™

(Protease)

MultiTox Fluor

MultiTox-Glo

Apo-ONE®

Caspase Assay

Caspase-Glo®

ApoLive-Glo™

CellTiter-Blue®

•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

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

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

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

ATP Assay for Cell Viability

Dead Cell

Viable Cell

Light No Reaction

Luciferin + Luciferase

CellTiter-Glo Reagent

• Lysis Solution • ATPase Inhibitors • Luciferin • UltraGlo Luciferase

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

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

22⁰C 4⁰C

CellTiter-Glo® 12 hours 3.5 days

new reagent 2 weeks 4 months*

10% change in performance

Balb 3T3 Cells Treated with MTT for 4 Hours

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.

Balb 3T3 Cells Treated with GF-AFC for 4 Hours

Detecting Dead Cells: Two Basic Approaches

Viable Dead

The functional definition of cell viability is based on whether the

outer membrane is intact.

Dye Enzyme Marker

• Protease marker

• ATP content

• NADH

DNA Dye Staining to Detect Dead Cells (Overcomes some limitations of short half-life markers)

Dye is excluded from live cells

DNA dye only stains nucleus of “dead” cells or debris

Non-permeable

DNA dye

Staining of dead

cells results in a

fluorescent signal

that is stable. X

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

HeLa Cells Treated with CellTox-Green DNA Dye for 4 Hours

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?)

Samples with CellTox Green can be Multiplexed with Cell Viability and Apoptosis Assays

Triplex at each time point Endpoint multiplexes possible at first emergence of toxicity

24hr 48hr

Note progression of cytotoxicity and gradual decline in caspase activity suggesting loss of enzymatic activity after release from dead cells.

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

CellTox™ Green Assay: Multiplexing a Fluorescent Assay with Luminescent Assays

CellTiter-Fluor™ Viability

CellTox Green™ Cytotoxicity

BacTiter-Glo™ Assay

CellTiter-Glo® Cell Viability

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

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®

Assays to Determine Cell Stress Events Leading to Toxicity

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

• Metabolic markers (NADH, NADPH)

• Gene expression in several stress-related pathways

Detecting Apoptosis as the Mechanism of Cell Death

• Protease marker

• ATP content

• NADH

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

H N O Z - D E V D

H N N H D V E D - Z Z - D E V D

C a s p a s e 3

F l u o r e s c e n c e

H 2 N O

H 2 N N H 2

C a s p a s e 3

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

H 2 N C O O

AMC, R110 and aminoluciferin substrates for measuring caspase activity

Luminescent Caspase Assay

Caspase-Glo® Reagent

Reagent No Rxn X

Apoptotic Cell

Reagent Luminescence

• Lysis Solution

• Z-DEVD-aminoluciferin

• Stable Luciferase

• ATP

Pro-Caspase Inactive

Inactive Caspase

Active Caspase

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 )

(Thousands)

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

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

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

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.

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

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

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)

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

Principal of GSH:GSSG Ratio Assay (Assays must be run in parallel in separate wells.)

Total Glutathione

Reduce With DTT

Oxidized GSSG

Block with NEM

GSSG GSH Reduce

Luciferase, ATP (LDR)

Menadione Treatment Drops GSH:GSSG Ratio

GSH:GSSG changes indicate: • Oxidative Stress • Compound toxicity • Reactive metabolite formation

Reactive Oxygen Species (ROS) Assay

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

ROS-Glo Assay Chemistry Based on Pro-Luciferin

• Self-cleaving linker

• D-Cys cyclization

Luciferase

Modified Pro-luciferin

ROS-Glo™ H2O2 Assay Protocol

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

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

Assays for NADH/NADPH, NAD+, & NADP Metabolic Indicators

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

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

Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides

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

Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides

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

Bioluminescent Assay Approach is More Sensitive

Summary of NAD(P) / NAD(P)H Assays

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

Cell Stress Response Pathway Reporters

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.

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.

Skin Sensitization Model Pathway KeratinoSens Cell Line from Givaudan

Modified from Fig. 2 from Natsch, A. Tox. Sci. 113(2), 284–292 (2010)

Stress and Toxicity Pathway Vectors

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]

Cell lines available as custom products http://www.promega.com/a/forms/custom-assays/custom-assay-services.html

Questions Welcome

March, 2013 Terry.Riss@Promega.com

cell-based assays to detect the mechanism of toxicity/media/files/promega worldwide/europe/promega...

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