New Techniques for Continuous Real Time Monitoring of Viability and Cytotoxicity

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1 New Techniques for Continuous Real Time Monitoring of Viability and Cytotoxicity Zhong Yu, PhD Product Manager BeNeLux & Nordic March

2 Presentation Outline Measuring viable cell number in real time How the assay works Multiplexing Advantages & Disadvantages Measuring accumulation of dead cells in real time How the assay works Multiplexing Advantages & Disadvantages Summary Lab experiment: How healthy are your cells?

3 Metabolic Indicators of Cell Viability Reagent Tetrazolium Reagents MTT, MTS, XTT Redox Indicators Resazurin Enzyme Substrates Protease Substrates Viable Cell Active Metabolism Dead Cell X Substrate Product Substrate No Rxn Incubation Period 3

4 Balb 3T3 Cells Treated with MTT for 4 Hours Time Zero 4 hours Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences Assay Guidance Manual:

5 Balb 3T3 Cells Treated with Resazurin for 4 Hours Time Zero 4 hours Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences Assay Guidance Manual:

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

7 Real Time Cell Viability Assay

8 RealTime-Glo MT Cell Viability Assay Measures Reducing Potential of Cells Pro-substrate Live cell Pro-substrate Dead cell Luciferase Pro-substrate Intracellular Reduction Substrate Pro-substrate X Substrate Light No Signal Luciferase and Pro-substrate are added as reagents to culture medium Pro-substrate enters the cell and is reduced to form a substrate for luciferase Substrate diffuses from the cell and is used by luciferasse to produce light

9 RealTime-Glo Assay Protocol Seed cells in medium containing RealTime-Glo Reagent Add test compound Record luminescence (continually for up to 3 days)

10 Measure Changes in Viability Over Time The luminescence signal was determined every hour for 72 h in a Tecan M200 plate reader with gas control module (37C/5%CO 2 )

11 Luminescent Signal Drops Immediately Upon Cell Death Add digitonin icell cardiomyocytes were plated and grown in medium containing pro-substrate and NanoLuc luciferase. After 2 days, digitonin was added to a final concentration of 200 µg/ml

12 Comparison of RealTime-Glo Assay to alamarblue Endpoint Approach RealTime-Glo Assay is more sensitive, rapid, and robust than resazurin A549 cells were treated with digitonin for 24 h. Viability reagents were added and signal read at 30 min post-addition (RealTime-Glo Assay) or 4 hours post-addition (alamarblue)

13 Multiplexing Real Time Cell Viability Assays

14 ONE-Glo (RLU) RealTime-Glo (RLU) Multiplexing Viability and Luciferase Reporter Assays Seed HEK293 cells expressing luciferase in 384 well plate 2.00E E+06 ONE-Glo multiplex ONE-Glo alone 5.00E E+04 Incubate overnight 1.20E E+04 Add RealTime-Glo Reagent 8.00E E+04 Incubate 2 hours 4.00E E+04 Record Luminescence Add firefly luciferase reagent 0.00E E cells/well Incubate 10min Record luminescence Firefly luciferase reporter assay signal is not affected by the presence (red squares) or absence (green triangles) of RealTime-Glo Reagent

15 Signal/Background RealTime-Glo MT Cell Viability Assay Applied to 3D Microtissues Hanging Drop Spheroids of HEK293 Cells Microtissue Diameter (µm)

16 Yield (ng) RealTime-Glo Reagents Do Not Effect RNA Yield using QuantiFluor RNA System 1000 RNA yield Maxwell + RT-Glo Maxwell - RT-Glo ReliaPrep + RT-Glo ReliaPrep - RT-Glo Microtissue Diameter (µm) Proprietary Information. Not for further distribution. 20

17 RealTime-Glo Reagent Does Not Affect Cycle Threshold of Extracted RNA

18 Advantages of RealTime-Glo Assay Provides kinetic information on viable cell number during the course of experiments Viable cells remain after applying RealTime-Glo Reagent (i.e. the reagent is not toxic), allowing multiplex options. Optional protocols enable reagent to be added when cells are plated, when test compound is added, or at any time point when cell viability measurements are needed. Better sensitivity than colorimetric or fluorometric viability assays that measure reducing potential of cells

19 Real Time Detection of Dead Cells

20 CellTox Green Cytotoxicity Assay Real Time Method to Detect Dead Cells

21 DNA Dye Staining to Detect Dead Cells (Overcomes some limitations of short half-life markers) Non-permeable DNA dye CellTox Green 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 26

22 CellTox Green Dye Measures Accumulation of Dead Cells in Culture HepG2 cells were treated with various doses of Terfenadine. CellTox Green Dye was added and fluorescence was measured every hour for 3 days. Increasing fluorescence indicates an increase in the number of dead cells

23 CellTox Green Dye is Not Toxic to Cells and does not affect response to other toxins ATP assay data showing viability of cells exposed to DNA binding dye for 15 minutes or 72 hours. Dye is non-toxic for at least 72 hours No effect on IC 50 value of test compounds

24 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

25 Samples Stained with DNA Dye can be Multiplexed with Cell Viability and Apoptosis Assays Add DNA dye when seeding cells 24hr Incubate Record fluorescence from dead cells Add GF-AFC Reagent Record fluorescence from live cells Add Caspase Reagent Record luminescence from apoptotic cells 30

26 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

27 Advantages of DNA Staining Advantages: Real Time DNA staining of dead cells produces a fluorescent signal that lasts much longer than the signal from enzyme release. DNA staining dye overcomes the major disadvantage of enzyme release assays. Numerous multiplex opportunities because cells remain viable Disadvantage: Signal window is not as great as enzyme marker assays which can be amplified by enzymatic generation of product

28 Cell-based Assays to detect the mechanism of toxicity Apoptosis detected using Caspase activities Biochemical markers of cell stress leading to cytotoxicity Oxidative stress (ROS and GSH:GSSG) NAD/NADH, NADP/NADPH Metabolites (Lactate, Glutamate, Glucose) Luciferase reporters of cell stress pathways leading to cytotoxicity

29 Summary A novel assay has been developed to measure viable cell number in real time: Repeated kinetic luminescent measurements indicate cell growth or death over time Reagents are not toxic, thus cells remain viable for subsequent multiplexing assays A non-toxic non-permeable DNA dye can measure dead cell number in real time: Repeated fluorescence measurements indicate appearance of dead cells DNA dye is non-toxic, thus cells remain viable for subsequent multiplexing assays Real time detection methods provide flexibility during assay development: Kinetic measurements of cell health from the same plate eliminates the need for multiple parallel plates during development and optimization of phenotypic assays Multiplexing real time assay methods can provide an internal control to verify viable cell number simultaneously with a variety of other phenotypic assays

30 2015. Questions?