Multiplexing Cell-Based Assays: Get More Biologically Relevant Data

Transcription

1 Multiplexing Cell-Based Assays: Get More Biologically Relevant Data Kyle Hooper, PhD December 2011 Promega Webinar

2 Multiplexing assays for more informative data Introduction Plate-based assays for viability, cytotoxicity and apoptosis measurement Using multiplex assays to understand cell death mechanism Monitoring cell response in multiple applications

3 Definitions viable cells 1 necrosis apoptosis with 2 necrosis Cell Viability Assay Assays based on measuring a metabolic marker or cytoplasmic enzyme in cells with intact cell membranes. Assays could be lytic or non-lytic. Cytotoxicity Assay Assays based on cells that do not have intact cell membranes leaking normally cytoplasmic enzymes into the cell culture medium. Assays are non-lytic. Apoptosis Assay Assays based on measuring activation of specific caspases. Cells with activated caspase-3/7 are considered committed to apoptotic cell death. Assays are lytic.

4 Multiplexing is Gathering more than one set of data from the same sample Multiplexing requirements: Assays must be biologically & chemically compatible Signals must be spectrally distinct Assays must fit in the available volume of the well Assay #1 Assay #2

5 Why multiplex? To gain a more complete picture of what is happening to the cell Cells are apparently losing viability

6 Why multiplex? To gain a more complete picture of what is happening to the cell Cells are apparently losing viability but the membranes are intact

7 Why multiplex? To gain a more complete picture of what is happening to the cell Cells are apparently losing viability but the membranes are intact and caspase-3/7 has been activated therefore, cytostasis with early stage apoptosis!

8 Why multiplex? To gain a more complete picture of what is happening to the cell Cells are apparently losing viability but the membranes are intact and caspase-3/7 has been activated therefore, cytostasis with early stage apoptosis! Reduce faulty interpretation or ambiguity Eliminates variables from replicate plates Normalize data Increase data content per well, per compound aliquot

9 Plate-based assays for cell viability, cytotoxicity, and caspase-dependent apoptosis measurement

10 Major methods to assess cell viability Reducing Potential reduced MTT Soluble MTT reduced MTT insoluble Reduced Indicator Compound Resazurin ETR Resorufin ETR -reduced Indicator Compound MTS XTT WST

11 Major methods to assess cell viability Luciferin & Luciferase ATP Light Reducing Potential reduced MTT Soluble MTT reduced MTT insoluble Reduced Indicator Compound Resazurin ETR Resorufin ETR -reduced Indicator Compound MTS XTT WST

12 Major methods to assess cell viability Luciferin & Luciferase ATP Light profluorescent compound Enzyme Markers Esterases Live Cell Protease Reducing Potential reduced MTT Soluble MTT reduced MTT insoluble Reduced Indicator Compound Resazurin ETR fluorescent compound Resorufin ETR -reduced Indicator Compound MTS XTT WST

13 Cell Viability Assays Methods to measure live cells Product Measures Steps Time to results Sensitivity Method CellTiter-Glo Luminescent Cell ++++ ATP 3 10 minutes 10 cell Viability Assay (Luciferase) sensitivity Luminescent CellTiter-Fluor Cell Viability Assay (Gly-Phe-AFC) Live Cell Protease 3 30 minutes +++ Fluorescent Resazurin/Resorufin (fluorescent) e.g., CellTiter-Blue Cell Viability Assay Reducing potential hours ++± Fluorescent Soluble Formazan with Electron Transfer Reagent (MTS/XTT/WTS) e.g., CellTiter 96 AQ ueous One Reducing potential hours ++ Colorimetric Insoluble Formazan MTT e.g., CellTiter 96 Assay 3 H-thymidine incorporation assay Reducing potential hours ++ Colorimetric DNA Synthesis hours +++± Radiometric

14 Cytotoxicity Assays Lactate Dehydrogenase Lactate + NAD + INT Formazan -or- Diaphorase Pyruvate + NADH INT -or- Resazurin Assays are non-lytic

15 Cytotoxicity Assays Lactate Dehydrogenase Other enzyme markers of cytotoxicity: Dead Cell Protease Lactate + NAD + Pyruvate + NADH Adenylate Kinase Glyceraldehyde- 3-PO 4 -Dehydrogenase INT Formazan -or- Diaphorase INT -or- Resazurin Assays are non-lytic

16 Major markers for cytotoxicity assays Marker Method Time Marker Half-Life in Media Direct Cell-Based Assay Lactate Dehydrogenase (e.g., CytoTox 96 Cytotoxicity Assay & CytoTox-ONE Membrane Integrity Assay Colorimetric (Formazan Chemistry) Fluorescent (Resazurin/Resorufin) 30 min. ~10hr No 10 min. Yes Dead Cell Protease (e.g., CytoTox-Fluor & CytoTox-Glo Cytotoxicity Assays) Fluorescent 30 min. ~8 hr Yes Luminescent 15 min. Yes Glyceraldehyde-3-PO 4 -Dehydrogenase Luminescent 5 min. ~4 hr Yes Adenylate Kinase Luminescent 5 min. ~3 hr No Sensitivity Luminescent > Fluorescent > Colorimetric Governs how soon you have to assay after the cytotoxic event

17 % Activity Dead-Cell Protease more like LDH LDH (Fluorescent) 50 Dead-Cell Protease 25 Adenylate Kinase (luminescent) Glyceraldehyde-3-PO 4 - Dehydrogenase (luminescent) Hours After Cytotoxic Event

18 Enough signal to differentiate 5-10% cytotoxicity Dead-Cell Protease Better signal:noise ratios allows detection of minor changes in toxicity. AK Mixture of live and sonicated cells Plate read 15 minutes after reagent addition

19 Enough signal to differentiate 5-10% cytotoxicity 5-10% alive (D ~10,000 RLU) Dead-Cell Protease Better signal:noise ratios allows detection of minor changes in toxicity. AK 5-10% dead (D ~10,000 RLU) Mixture of live and sonicated cells Plate read 15 minutes after reagent addition

20 Plate-based assays for caspase-dependent apoptosis Early Apoptosis Assays based on measuring activation of specific caspases. Cells with activated caspase-3/7 are considered committed to apoptotic cell death. Assays are lytic. Caspase-3/7 Committed Apoptosis Aminoluciferin UltraGlo Luciferase + ATP Rhodamine 110 Light

21 Luminescence is most sensitive

22 Using multiplex assays to understand cell death mechanism How a search for a Glo-Type cytotoxicity assay lead to a multiplexing assay with tremendous versatility

23 Development timeline for cell-based viability, cytotoxicity and apoptosis assays CytoTox 96 Assay (INT) CytoTox-ONE Assay (Resazurin/Resorufin) Apo-ONE Caspase Assay (bisdevd-r110) Caspase-Glo 3/7 Assay (DEVD- NH 2 luciferin) MultiTox-Glo Assay (GF-AFC/AAF-NH 2 luciferin) MultiTox Fluor Assay (GF-AFC/AAF-R110) CytoTox-Glo Assay (AAF-Aminoluciferin) Caspase-Glo 8 & 9 Assay CytoTox-Fluor Assay (AAF-R110) CellTiter 96 Assay (MTT) CellTiter 96 AQ ueous Assay (PMS/MTS) ApoLive-Glo Multiplex Assay (GF-AFC / DEVD-Aminoluciferin CellTiter 96 AQ ueous ONE Soln (PES/MTS) CellTiter-Glo Assay (Luciferin/ Luciferase) CellTiter-Fluor Assay (GF-AFC) CellTiter-Blue Assay (Resazurin/ Resorufin) ApoTox-Glo Triplex Assay (GF-AFC/AAF-R110/ DEVD-Aminoluciferin

24 Development timeline for cell-based viability, cytotoxicity and apoptosis assays CellTiter 96 Assay (MTT) CytoTox 96 Assay (INT) CellTiter 96 AQ ueous Assay (PMS/MTS) CellTiter 96 AQ ueous ONE Soln (PES/MTS) CytoTox-ONE Assay (Resazurin/Resorufin) Apo-ONE Caspase Assay (bisdevd-r110) CellTiter-Glo Assay (Luciferin/ Luciferase) Caspase-Glo 3/7 Assay (DEVD- NH 2 luciferin) Caspase-Glo 8 & 9 Assay CytoTox-Fluor Assay (AAF-R110) CellTiter-Fluor Assay (GF-AFC) CellTiter-Blue Assay (Resazurin/ Resorufin) MultiTox-Glo Assay (GF-AFC/AAF-NH 2 luciferin) MultiTox Fluor Assay (GF-AFC/AAF-R110) Viability Assays MTT MTS Glo CytoTox-Glo Assay (AAF-Aminoluciferin) Apoptosis Assays TUNEL Ab s Extracts Cell-Based R110 Caspase-Glo 2, 6, 8 & 9 Assays (XXXX-Aminoluciferin) ApoTox-Glo Triplex Assay (GF-AFC/AAF-R110/ DEVD-Aminoluciferin Glo Cytotoxicity Assays INT Resazurin? ApoLive-Glo Multiplex Assay (GF-AFC / DEVD-Aminoluciferin

25 Two protease activities = live/dead cell assay Are my cells living? Are my cells dying? Live-Cell Protease Dead-Cell Protease Niles, A.L., et al. (2007) Analytical Biochemistry 366,

26 Measure Live, Dead or Both Live-Cell Substrate crosses the membrane GF-AFC AFC Live-Cell Protease Assay CellTiter-Fluor Cell Viability Assay Live-Cell Protease

27 Measure Live, Dead or Both Live-Cell Substrate crosses the membrane GF-AFC Live-Cell Protease AFC Live-Cell Protease quickly inactivated outside the cell Live-Cell Protease Assay CellTiter-Fluor Cell Viability Assay AFC Fluorescence is proportional to the number of live cells

28 Measure Live, Dead or Both Live-Cell Substrate crosses the membrane GF-AFC AFC Live-Cell Protease quickly inactivated outside the cell Live-Cell Protease Assay CellTiter-Fluor Cell Viability Assay Live-Cell Protease Dead-Cell Protease AAF-NH 2 -Luciferin bis-aaf-r110 Dead-Cell Protease Dead-Cell Protease Assay CytoTox-Fluor Cytotoxicity Assay CytoTox-Glo Cytotoxicity Assay Light Rhodamine 110 Compromised membranes allow Dead-Cell Protease access to the substrate

29 Measure Live, Dead or Both Live-Cell Substrate crosses the membrane AAF-NH 2 -Luciferin GF-AFC Live-Cell Protease Dead-Cell Protease bis-aaf-r110 AFC Dead-Cell Protease Substrates cannot cross intact membranes AAF-NH 2 -Luciferin Live-Cell Protease quickly inactivated outside the cell Light bis-aaf-r110 Dead-Cell Protease Rhodamine 110 Compromised membranes allow Dead-Cell Protease access to the substrate Live-Cell Protease Assay CellTiter-Fluor Cell Viability Assay Dead-Cell Protease Assay CytoTox-Fluor Cytotoxicity Assay CytoTox-Glo Cytotoxicity Assay R110 Fluorescence or Luminescence is proportional to the number of dead cells

30 Measure Live, Dead or Both Measure Live Cell Fluorescence GF-AFC Live-Cell Protease AFC Live-Cell Protease Assay CellTiter-Fluor Cell Viability Assay Dead-Cell Protease Assay CytoTox-Fluor Cytotoxicity Assay CytoTox-Glo Cytotoxicity Assay MultiTox-Fluor 1-step add-mix-measure/measure MultiTox-Glo 2-step A-M-M (AFC)/A-M-M (Glo) AAF-NH 2 -Luciferin Light bis-aaf-r110 Dead-Cell Protease Rhodamine 110 Measure Dead Cell Fluorescence or Luminescence Live- & Dead-Cell Assay MultiTox-Fluor Multiplex Cytotoxicity Assay MultiTox-Glo Multiplex Cytotoxicity Assay

31 Inverse relationship between live & dead cell signals Live Cells Raw or Signal to Noise Dead Cells Percentage of maximal signal

32 Ratiometric measures address variability MultiTox-Fluor Assay Data Single parameter assay can be affected by experimental variation 12K 11K 10K 9K 7.5K 5K Pipetting error or cell clumping can cause variation in seeding density, for example Ratiometric measure decreases variation by normalizing the data Mixture of 50% viable cells/50% lysed cells Variable amount added to wells

33 MultiTox-Fluor assay improves data confidence for cytotoxicity screens More cells/well Dead-Cell Protease Assay Fewer cells/well Live-Cell Protease Assay A cytotoxic event must yield an increase in dead-cell protease activity and a decrease in live-cell protease activity

34 MultiTox-Fluor can be the perfect multiplexing partner Multiplexing requirements: Assays must be biologically & chemically compatible Signals must be spectrally distinct Assays must fit in the available volume of the well Multi-Tox Fluor Non-Lytic 2 data points Lytic Luminescent Assay

35 Multiplexing with Caspase-Glo 3/7 Assay MultiTox-Fluor Multiplex Cytotoxicity Assay matches well with the Caspase-Glo 3/7 Assay Used as the multiplexing example in the MultiTox- Fluor manual Example paper Caspase-dependent apoptotic cell death Apolloni, S., et al. (2010) Neurochem. Intl. 56, An ideal cell health multiplex assay

36 Deciphering a complicated process: Multiplex assays -> Cytotoxicity signatures ApoTox-Glo Assay: Non-lytic Fluorescent Live Cell Assay 1 VIABLE ATP ATP ATP ATP ATP Live-Cell Protease Dead-Cell Protease ATP ATP ATP ATP ATP REDUCED VIABILITY ProCaspase-3 APOPTOSIS 1 CellTiter-Fluor Assay 2 CytoTox-Fluor Assay 3 Caspase-Glo 3/7 Assay ApoLive-Glo Assay: 1 CellTiter-Fluor Assay 3 Caspase-Glo 3/7 Assay AAF- -FAA Caspase-3 Rhodamine 110 NECROSIS 2 Non-Lytic Fluorescent Dead Cell Assay 3 Lytic Bioluminescent Caspase-3/7 Assay Light

37 Multiplexing addresses the Cytotoxicity Problem: A Simple Concept with Inherent Biological Complexity Did the treatment affect viability? Yes/No? How? When? How potent was the treatment? Is the treatment selective? The cytotoxic phenotype is shaped by multiple factors: 1. Dosage 2. Exposure Time 3. Cellular susceptibility No single parameter assay can fully characterize cytotoxicity

38 ApoTox-Glo Triplex gives signatures of cell status Each stage illustrated gives a characteristic response from all 3 assays The following slides use different: treatment compounds exposure times to give a moment in time that illustrates each stage and characteristic signature.

39 Signature #1. No Cytotoxic Effect DIC image courtesy of Chad Zimprich viable cells Compound dose & exposure time and cell type are critical parameter for establishing cellular inertness.

40 Signature #2. Primary Necrosis Necrotic cells Rapid loss of membrane integrity (<4hrs) without caspase activation is strongly indicative of primary necrosis.

41 Signature #3 Cell cycle arrest and early apoptosis DIC image courtesy of Chad Zimprich Decrease in apparent viability (viable cell number), no change in cytotoxicity, with increase in caspase activation are consistent with cell-cycle arrest.

42 Signature #4: Apoptosis DIC image by Chad Zimprich Decrease in viability with a commensurate increase in cytotoxicity and caspase activation are consistent with apoptosis and secondary necrosis

43 Signature #5: Late State Apoptosis Dose-dependent decrease in viability, increase in cytotoxicity & apoptosis, with caspase biomarker degradation at highest concentrations is consistent with late stage apoptosis.

44 Potency & Safety Evaluation: Validation of Clinical Cancer Therapeutics with ApoTox-Glo Assay icells are specifically designed to aid drug discovery and improve the predictability of drug efficacy and toxicity screens, weeding out ineffective and potentially toxic compounds early in the pharmaceutical pipeline process before significant time and resources have been invested. -Cellular Dynamics International icells Cardiomyocytes evaluate off-target effects FDA Approved Anti-Leukemia Drugs K562 Human Leukemic Cells evaluate on-target effects ApoTox-Glo (After 24hr Exposure)

45 HDAC inhibitor shows target specificity icell Cardiomycetes ApoTox-Glo K562 Leukemia cells ApoTox-Glo No apparent cytotoxicity or caspase activation. Cytotoxicity by apoptosis Histone Deacetylase Inhibitor SuberoylAnilide Hydroxamic Acid (Vorinostat )

46 Monitoring cell response in multiple applications How two fluorescent assays can augment bioluminescent assays

47 Understand overall cellular response to treatment Luciferase Reporter Assay Reporter activity goes down with increasing compound Is this real? Did the treatment adversely affect the cell?

48 Understand overall cellular response to treatment Luciferase Reporter Assay AFC LIVE-CELL PROTEASE GF-AFC CellTiter-Fluor Viability Assay A simple 30 minute assay upstream of the reporter assay can answer that question!

49 Understand overall cellular response to treatment Luciferase Reporter Assay AFC LIVE-CELL PROTEASE GF-AFC CellTiter-Fluor Viability Assay The decrease in reporter activity was due to the death of the cells, not down regulation of the promoter

50 Normalize Reporter Data to viable cells Raw Luminescence Bright-Glo Luciferase Assay Steady-Glo Luciferase Assay Normalized Raw Luminescence GloResponse NF-kB Cells 5K to 25K cells/well Reporter assay Raw RLU results -OR- Normalized to multiplexed CellTiter-Fluor Viability Assay Normalized

51 Monitor changes in GSH levels in cells under stress UltraGlo Luciferase + ATP Light A change in GSH levels is important in assessment of toxicological responses and is an indicator of oxidative stress, potentially leading to apoptosis or cell death. GSH-Glo Glutathione Assay

52 Multiplexing Live/Dead and GSH Level Assays Add MultiTox-Fluor Reagent Incubate 30 min Record Fluorescence Remove medium (GSH background) Add GSH-Glo Reagent,30min Add Luciferin Detection Reagent, 15min GSH Synthesis Inhibitor Again, timing is everything. In this window, there is no change in viability but be assured, a lack of GSH will lead to cytotoxicity. Record Luminescence

53 Histone Deacetylases are cancer targets Deacetylated histones have tight structure, restricting transcription Acetylated histones have a loose structure, encouraging transcription Balance of acetylation and deacetylation is important & an imbalance is not good. Hess-Stumpp et. al HDAC-Glo I/II Assay HDAC Developer Reagent UltraGlo Luciferase ATP Light HDAC-Glo I/II Substrate

54 Multiplexing Live/Dead and HDAC activity assays to identify on- and off-target effects icell U937 No apparent cytotoxicity Cytotoxicity Multiplexed measurement of HDAC inhibitor activity with HDAC-Glo I/II and cytotoxicity with Multitox-Fluor TM Reagent clearly delineates cell lines that are sensitive to HDAC inhibitors. Exposure of two cell lines to apicidin for 24h followed measurement identified the HDAC inhibitor sensitive cell line and showed good correlation between HDAC inhibition and cytotoxicity.

55 Conclusion

56 What multiplexing can do for you Multiplexing gives a more complete picture of what s happening in the cell Reduces ambiguity Eliminates variables Normalizes data Increases content

57 Viable Cell Determine Cytotoxicity/Cell Death Mechanism with ApoTox-Glo Triplex Assay ApoTox-Glo Triplex Assay measures: Live Cells Dead Cells Apoptotic Cells and gives profile signatures Live-Cell Protease Apoptosis 1 3 ProCaspase-3 Caspase-3 2 Necrosis Dead-Cell Protease 1 Necrosis 2

58 Live/Dead assays easily added upstream Incubate 30 minutes Treat cells in white or black wall plates. Add CellTiter-Fluor, CytoTox-Fluor, or MultiTox-Fluor Reagent ONE-Glo Reporter Assay Bright-Glo Reporter Assay Steady-Glo Reporter Assay Renilla-Glo Reporter Assay Beta-Glo Reporter Assay Caspase-Glo 3/7 Assay Caspase-Glo 8 Assay Caspase-Glo 9 Assay ApoONE Caspase-3/7 Assay* HDAC-Glo I/II Assay (coming soon) GSH-Glo Glutathione Assay P450-Glo Cell-Based Assays Measure AFC & R110 fluorescence Perform 2 nd assay using normal protocol CellTiter-Glo Assay CytoTox-ONE Assay

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