Cell Health and Mechanistic Toxicity Assays

Transcription

1 Cell Health and Mechanistic Toxicity Assays Zhong Yu, PhD Product Manager BeNeLux & Nordic March 2015

2 Outline Bioluminescent assays Live and Dead Cell Assays Apoptosis Stress Events Leading to Cytotoxicity 2

3 Bioluminescent Assays Developed From Firefly Luciferase Chemistry Reporter gene assays Cell Viability Kinase assays camp & PDE assays P-glycoprotein assay Caspases/proteases CYP450 assays HDAC assays GSH or ROS assays NAP(D) (H) assays New N- & C- termini N C Fuse wt N- & C- termini GloSensor camp GloSensor caspase

4 Cell Health Assays Overview Viable cells detected using markers of active metabolism Cellular conversion of indicator dyes (MTT / MTS / Resazurin) Protease marker ATP content Real time viability assay using NanoLuc 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 noxidative stress (ROS and GSH:GSSG ratio) NADH Luciferase reporters of cell stress pathways leading to cytotoxicity 4

5 Metabolic & Enzymatic Indicators of Cell Viability Reagent Tetrazolium Reagents MTT, MTS, XTT, WST Redox Indicators Resazurin Enzyme Substrates Protease Substrates GF-AFC Viable Cell Active Metabolism or Protease Dead Cell Loss of Function Substrate Incubation Step Product Substrate X No Rxn

6 Balb 3T3 Cells Treated with MTT for 4 Hours Same field of cells imaged immediately after addition of MTT and after 4 hours incubation. Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences. 6

7 Balb 3T3 Cells Treated with Resazurin for 4 Hours Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences. 7

8 Balb 3T3 Cells Treated with GF-AFC for 4 Hours 4 h exposure used for comparison; but 30 min is usually adequate CellTiter-Fluor Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences. 8

9 ATP Assay for Cell Viability (immediate lysis) CellTiter-Glo 2.0 Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell Dead Cell ATP Light Luciferin + Luciferase ADP X No Reaction

10 Advantages & Disadvantages of Viability Assays Assay Assay Disadvantages MTT / MTS Widely used Inexpensive 1-4 hour incubation Interference by reducing compounds Toxic to cells Limited sensitivity (absorbance) CellTiter 96 AQ ueous One Solution Cell Proliferation Assay Resazurin Protease ATP Inexpensive Fluorescent readout Good sensitivity CellTiter-Blue Cell Viability Assay 30 min protocol Cells remain viable Better sensitivity than resazurin Good choice for multiplexing CellTiter-Fluor Cell Viability Assay 10 min protocol Best sensitivity No fluorescence interference Lysis step stops reaction immediately (no incubation with viable cells) 1-4 hour incubation Interference by reducing compounds Toxic to cells Fluorescence interference Fluorescence interference Lytic protocol dictates sequence for multiplexing CellTiter-Glo 2.0 Luminescent Cell Viability Assay 10

11 RealTime-Glo MT Cell Viability Assay Based on NanoLuc Luciferase

12 Real Time Cell Viability Assay Measures Reducing Potential of the Cell Live Cell Dead Cell NanoLuc protein sensor is present outside of the cells Pro-furimazine substrate enters the cell and is reduced by the cell to form furimazine Furimazine (substrate) diffuses from the cell and is rapidly used by NanoLuc to produce light 12

13 Detecting Dead Cells: Two Basic Approaches The definition of cell viability is based on membrane integrity. Live Dead Apoptotic Dye Enzyme Marker

14 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

15 Enzyme Marker Release Assay to Detect Dead Cells Detection Reagent CytoTox-ONE Viable Cell Dead Cell Enzymes retained in live cells X No Reaction Fluorescence 16

16 F l uorescence ( Thousands) Stability of released enzyme activity in culture medium becomes a limitation 12 L DH-Re le a s e As s a y T im e Co u rs e Tam oxif en Tr eat ed HepG 2 Cels CytoTox-ONE 10 0hr hr 6 4 2hr 6hr 24hr See: Assay & Drug Devel Tech 2(1): 51, 2004 T a m o x i fe n (µ M ) LDH activity in medium decreases after 24 hours A A

17 Dead Cell Protease Assay (Fluorescent) Impermeable Protease Substrate CytoTox Fluor Dead Cell protease remains active long after cell death Only signal is from Dead Cells Impermeable substrate cannot enter viable cells Viable Cell X bis-aaf-r110 No Signal from Viable Cells Dead Cell Dead Cell Protease Active Dead Cell Protease bis-aaf-r110 R110 Fluorescence 485/520nm

18 Viable Cell Protease Assay (Fluorescent) Cell Permeable Protease Substrate CellTiter Fluor Viable cells retain protease activity and generate signal Viable Cell protease becomes inactive upon cell death GF-AFC Viable Cell Viable Protease AFC Fluorescence 400/505nm Dead Cell Viable Protease Inactive Inactive Viable Protease

19 Fluor escence ( Viable) ( Thousands) Fluor escence ( Dead) Multiplexing Measurement of Viable Cells & Dead Cells Simultaneously Subjected cells to various treatments 6000 M u ltip le x As s a y o f Via b le a n d De a d Ce lls b y M e a s u rin g Pro te a s e Ac tiv itie s Add Reagent with both Substrates Incubate min Record Fluorescence at 2 wavelengths I onom ycin ( µm ) Viable Dead 0. 00

20 DNA Dye Staining to Detect Dead Cells in real-time (Overcomes some limitations of short half-life markers) CellTox Green Non-permeable DNA dye Staining of dead cells results in a fluorescent signal that is stable. Viable Cell X Dead Cell Dye is excluded from live cells DNA dye only stains nucleus of dead cells or debris 21

21 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?)

22 Samples with CellTox Green can be Multiplexed with Cell Viability and Apoptosis Assays 24hr

23 Real Time Viability and Cytotoxicity Cytotoxicity Viability MCF7 cells (500 cells/well) dosed with etoposide with 2x Real Time Glo Cell Viability reagents, and 2x CellTox Green in media. Fluorescence and luminescence measured on Tecan M200 with Gas Control Module (37C/5% CO2) every 1 h for 72 h.

24 Advantages & disadvantages of assays to detect dead cells Assay Advantages Disadvantages LDH release Widely used and accepted Limited sensitivity CytoTox-ONE Homogeneous Membrane Integrity Assay Absorbance or fluorescent options 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 CytoTox-Fluor Cytotoxicity Assay 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 CellTox Green Cytotoxicity Assay Fluorescence interference Less sensitive than amplified protease release assay 25

25 Assays to Determine Cell Stress Events Leading to Toxicity

26 Determining Mechanisms Leading to Cytotoxicity Going beyond the standard assays available to detect live or dead cells. Assay chemistries and approaches to detect Apoptosis Oxidative stress (ROS and GSH:GSSG ratio) NAD/NADH, NADP/NADPH Mitochondrial toxicity Genetic reporters to detect stress response pathways 27

27 Detecting Apoptosis as the Mechanism of Cell Death

28 Luminescent Caspase-Glo 3/7 Assay Coupled enzyme assays enzyme of interest releases luciferin peptide, carbohydrate, or small molecule 31

29 Luminescent Caspase Assay Caspase-Glo Reagent Lysis Solution Z-DEVD-aminoluciferin Stable Luciferase ATP Viable Cell Apoptotic Cell Dead Cell Pro-Caspase Inactive Active Caspase Inactive Caspase Reagent X No Rxn Reagent Luminescence Reagent X No Rxn

30 Lumi nescence ( Thousands) Caspase-Glo 3/7 Time Course Indicates Caspase Activity is Transient Tamoxifen Treatment of HepG2 Cells Caspase Act ivit y Cells are apoptotic at 1 hr treatment with 100µM Tamox 24 hr 6 hr 4 hr hr 1 hr hr Assay & Drug Devel Tech 2(1): 51, T a m o x i fe n (µ M ) Caspase activity decreases after 24 hours incubation

31 Luminescent Caspase-3/7 Assay Advantages: Homogeneous (add-mix-measure) X 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

32 Oxidative Stress Assays

33 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: ROS (super oxide, hydroxyl radical, nitric oxide, hypochlorite convert to more stable H 2 O 2 ) Altered GSH:GSSG ratio (lowered GSH, increased GSSG) 37

34 ROS-Glo Assay Chemistry Based on Pro-Luciferin Modified Pro-luciferin (Peroxide Sensor) H 2 O 2 LDR (luciferase & ATP) Self-cleaving linker leaves D-Cys cyclization occurs Luciferase Light

35 ROS-Glo H 2 O 2 Assay Protocol Add test compound and modified pro-luciferin peroxide detector Incubate up to 2 hours ROS-Glo H 2 O 2 Assay of Hep G2 Cells Treated with Menadione Add luciferin detection reagent Incubate 15 min Record luminescence 39

36 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 40

37 Principal of GSH:GSSG Ratio Assay (Assays must be run in parallel in separate wells) Total Glutathione O N S N S COOH Reduce With DTT GSSG O S O NO 2 GSH GSH CH 3 GST GS-R GSH Block with NEM GSSG Reduce GSH HO N S N S COOH Luciferase, ATP (LDR) Oxidized GSSG Light 41

38 Menadione Treatment Drops GSH:GSSG Ratio GSH:GSSG changes indicate: Oxidative Stress Compound toxicity Reactive metabolite formation

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

40 Bioluminescent Assays for Adenine Dinucleotides Bioluminescent assays are available: 1. NADH + NADPH (total of both reduced forms) 2. NAD + + NADH (total non-phosphorylated) 3. NADP + NADPH (total non-phosphorylated) 45

41 Basic Principle for the Bioluminescent Adenine Dinucleotide Detection Assays 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 NAD(P)H NA(D)P + Diaphorase Modified Luciferin Luciferin

42 Cycling Enzymes and Corresponding Substrates Provide Selectivity for Measuring Nucleotides Glucose-6-phosphate and G-6-PDH are used to measure total phosphorylated NADP + NADPH Lactate dehydrogenase and lactate are used to measure total nonphosphorylated NAD + + NADH Product G-6-P Product Lactate G-6-PDH LDH NADPH NADP + NADH NAD + Diaphorase Diaphorase Proluciferin Luciferin Proluciferin Luciferin 47

43 Bioluminescent Assay Approach is More Sensitive 49

44 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 50

45 Summary Bioluminescent assays Live and Dead Cell Assays Apoptosis Stress Events Leading to Cytotoxicity 52

46 Questions Welcome