Multiplexing Cell-Based Assays:

Transcription

1 Multiplexing Cell-Based Assays: Get More Biologically Relevant Data Karthik Narasimhan, PhD Field Application Specialist, Asia Pacific Promega Corporation 2010, Promega Corporation.

2 Multiplexing assays for more informative data 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 cytoplasmic enzyme or marker 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 Assays must be chemically & biologically compatible Signals must be spectrally distinct Assay chemistries must be compatible The two assays must fit in the available volume of the well or be separable. Assay #1 Assay #2

5 What is the motivation for multiplexing? A more complete picture of what is happening to the cell. Cells are apparently losing viability but the membranes are intact. How? 1. Reduce faulty interpretation or ambiguity from data sets 2. Eliminate variables from culturing duplicate or triplicate plates Apparent loss of viability, intact membranes, caspase-3/7 activation therefore, cytostasis with early stage apoptosis! 3. Normalize data 4. Increase the content

6 2010, Promega Corporation. Plate-based assays for cell viability, cytotoxicity, and caspase-dependent apoptosis measurement

7 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) Caspase-Glo 8 & 9 Assay MultiTox-Glo Assay (GF-AFC/AAF-NH 2 luciferin) MultiTox Fluor Assay (GF-AFC/AAF-R110) CytoTox-Glo Assay (AAF-Aminoluciferin) CytoTox-Fluor Assay (AAF-R110) Caspase-Glo 8 & 9 Assays (XXXX-Aminoluciferin) 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

8 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

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

10 Cytotoxicity Assays Assays are non-lytic. Lactate Dehydrogenase Other Enzymes for luminescent output: Dead Cell Protease Lactate + NAD + Pyruvate + NADH Adenylate Kinase Glyceraldehyde- 3-PO 4 -Dehydrogenase INT -or- Resazurin INT Formazan -or- Diaphorase

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

12 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

13 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 Substrate Committed Apoptosis Rhodamine 110 Aminoluciferin UltraGlo Luciferase + ATP Caspase-3/7 Light

14 Luminescence is most sensitive

15 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 2010, Promega Corporation.

16 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

17 Other enzymatic markers of cytotoxicity 2 ADP Adenylate Kinase NADH GAPDH NAD + + P i Assay performed on conditioned media only 1 ATP + 1 AMP Luciferin/ Luciferase Glyc-1,3-diPO 4 Glyc-3-PO 4 PGK Light ADP ATP Luciferin/ Luciferase Light

18 Luminescent methods where not ideal LDH (Fluorescent) % Activity Adenylate Kinase (luminescent) Glyceraldehyde-3-PO 4 -Dehydrogenase (luminescent) Hours After Cytotoxic Event

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

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

21 Measure Live, Dead or Both Live-Cell Substrate crosses the membrane GF-AFC Live-Cell Protease Assay CellTiter-Fluor Cell Viability Assay Live-Cell Protease AFC Dead-Cell Protease Assay CytoTox-Fluor Cytotoxicity Assay CytoTox-Glo Cytotoxicity Assay bis-aaf-r110 AAF-NH 2 -Luciferin AAF-NH 2 -Luciferin bis-aaf-r110 Dead-Cell Protease Live- & Dead-Cell Assay MultiTox-Fluor Multiplex Cytotoxicity Assay Dead-Cell Protease Substrates cannot cross intact membranes Light Rhodamine 110 MultiTox-Glo Multiplex Cytotoxicity Assay Compromised membranes allow Dead-Cell Protease access to the substrate

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

23 Ratiometric measures address variability MultiTox-Fluor Assay Data Single parameter responses are partially dependent on cell number 12K 11K 10K 9K 7.5K 5K Subtle clumping or pipetting error can make screens difficult to interpret Ratiometric measures decrease variation by normalizing the data Variable number of 50% viability cells plated per well.

24 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

25 MultiTox-Fluor can be the perfect multiplexing partner Assays must be chemically & biologically compatible Signals must be spectrally distinct (Fluorescence or Luminescence) Assay chemistries must be compatible The assays must fit in the available volume of the well or be separable. Multi-Tox Fluor Non-Lytic 2 data points Lytic Luminescent Assay

26 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 Caspase-dependent apoptotic cell death This combination can do so much more

27 The Cytotoxicity Paradox: A Simple Concept with Inherent Biological Complexity Did the treatment affect cell 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

28 Deciphering a complicated process: Multiplexed, 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

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

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

31 Signature #3 Cell Cycle Arrest and early apoptosis DIC image courtesy of Chad Zimprich Decreases in apparent viability (viable cell number) with increases in caspase activation are consistent with cell-cycle arrest.

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

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

34 Does Biological Relevance Equate into Translational Relevance? Translational Problem: Patients with [various cancers] experience poor outcomes, especially in metastasized disease, and treatment of all stages is associated with strong side effects [off-target] resulting in impaired quality of life. Specific therapies for such high-risk patients are therefore urgently needed to resolve this unsatisfactory situation. Milde, T., et al. (2010) Clinical Cancer Research 16,

35 Potency & Safety Evaluation: Validation of ApoTox- Glo Assay with Clinical Cancer Therapeutics 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)

36 HDAC inhibition shows target specificity icell K562 No apparent cytotoxicity or caspase activation. Cytotoxicity by apoptosis Histone Deacetylase Inhibitor SuberoylAnilide Hydroxamic Acid (Vorinostat )

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

38 Monitoring cell response in multiple applications How two fluorescent assays can help bioluminescent assays 2010, Promega Corporation.

39 Distinct enzyme chemistries allow multiplexing in the Dual-Luciferase Assay Renilla Luciferase levels vary little with treatment The Stop & Glo Reagents quench firefly luminescence and provide the substrate for Renilla luciferase Mix 20µl of cell lysate and 100µl of Luciferase Assay Reagent II in the tube. Measure the light produced by firefly luciferase. Add 100µl of Stop & Glo Reagent to the tube. Control Renilla corrects for: cell death bad transfection Measure the light produced by Renilla Luciferase.

40 Understand overall cellular response to treatment LIVE-CELL PROTEASE AFC GF-AFC If only reporter activity is measured, the impact of compound treatment on cell health may be overlooked.

41 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

42 Normalize Reporter Data to viable cells Raw Luminescence Bright-Glo Luciferase Assay Normalized GloResponse NF-κB Cells at 5K to 25K cells/well Raw Luminescence Steady-Glo Luciferase Assay Normalized

43 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.

44 Multiplexing Viability and GSH Levels 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

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

46 Help identifying 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.

47 2010, Promega Corporation. Conclusion

48 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

49 Monitoring the cellular response improves data ApoTox-Glo Triplex Assay MultiTox-Fluor Multiplex Cytotoxicity Assay CytoTox-Fluor Cytotoxicity Assay CellTiter-Fluor Cell Viability Assay ApoLive-Glo Multiplex Assay 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 CellTiter-Glo Assay CytoTox-ONE Assay * works with CellTiter-Fluor Assay only Cell health monitoring is easily added upstream of many Promega cell-based assays Other multiplexing possibilities exist. Ask Technical Services

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