Novel Methods to Measure Cell Viability in Real Time Enables Multiplexing

Novel Methods to Measure Cell Viability in Real Time Enables Multiplexing ELRIG Nottingham March 18, 2015 2015

I don t want to hear any of this! 2015. 2

Presentation Outline What s wrong with existing assay technologies? 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 2015. 4

Metabolic Indicators of Cell Viability Reagent Tetrazolium Reagents MTT, MTS, XTT Redox Indicators Resazurin Enzyme Substrates Protease Substrates Viable Cell Active Metabolism Dead Cell 2015. X Substrate Product Substrate No Rxn Incubation Period 5

Why Not Use MTT or Resazurin Assays? They are endpoint assays MTT requires extra protocol steps to add reagent, return to incubator, add solubilization solution Sensitivity of MTT assay is limiting (~1000 cells) Interfering compounds: https://www.promega.com/resources/pubhub/isyour-mtt-assay-really-the-best-choice/ MTT and resazurin are toxic to cells (Assay Guidance Manual): http://www.ncbi.nlm.nih.gov/books/nbk144065/) 2015. 6

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: http://www.ncbi.nlm.nih.gov/books/nbk144065/ 2015. 7

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: http://www.ncbi.nlm.nih.gov/books/nbk144065/ 2015. 8

ATP Assay for Cell Viability (Endpoint Assay) Assay Reagent Lysis Solution ATPase Inhibitors Luciferin Stable Luciferase Viable Cell Dead Cell ATP ADP Light Luciferin + Luciferase X No Reaction

Real Time Cell Viability Assay 2015. 11

RealTime-Glo MT Cell Viability Assay Monitor viable cells continuously over 72 hours, saving time, cell samples, culture and reagent costs Option to add reagent at seeding, treating with compound, or at end Sensitivity is greater than colorimetric or fluorometric cell viability assays Multiplex with other assays and downstream applications 2015. 12

The RealTime Viability Assay Measures Reducing Potential of Cells 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 luciferase to produce light

RealTime Assay Protocol Seed cells in medium containing RealTime-Glo Reagent Add test compound and incubate Record luminescence (continually for up to 3 days) 2015. 14

Measure Changes in Viability Over Time Dose-response of Thapsigargin on A549 cells The luminescence signal was determined every hour for 72 h in a Tecan M200 plate reader with gas control module (37C/5%CO 2 ). 2015. 15

Luminescent Signal Drops Immediately Upon Cell Death digitonin Non-dividing Hepatocytes 2015. 16

RealTime-Glo Reagents are Not Toxic Membrane Integrity Assay PC3 or SKBR3 cells cultured in the presence or absence or RealTime-Glo Reagent for 3 days. Samples were tested for membrane integrity using CytoTox- Fluor Cytotoxicity Assay. 2015. 17

Comparison of RealTime-Glo Assay to alamarblue Endpoint Approach Resorufin from alamarblue assay accumulates in medium, so signal remains after cell death. Substrate from real time assay is rapidly used by NanoLuc, thus no accumulation. Add Reagents Incubate 2h Record Signals Add 1% Triton Incubate 1h Record Signals 2015. Minutes after 1% Triton addition Minutes after reagent addition 0 2 30 60 120 122 150 180 18

Multiplexing Real Time Cell Viability Assays 2015. 20

ONE-Glo (RLU) RealTime-Glo (RLU) Multiplexing RealTime Viability and Luciferase Reporter Assays Seed HEK293 cells expressing luciferase in 384 well plate 2.00E+06 1.60E+06 ONE-Glo multiplex ONE-Glo alone 5.00E+04 4.00E+04 Incubate overnight 1.20E+06 3.00E+04 Add RealTime-Glo Reagent 8.00E+05 2.00E+04 Incubate 2 hours 4.00E+05 1.00E+04 Record Luminescence Add firefly luciferase reagent (lysis occurs) 0.00E+00 0.00E+00 0 1000 2000 3000 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 2015. 21

Signal/Background RealTime-Glo MT Cell Viability Assay Applied to 3D Cell Spheroids 100 90 80 70 60 50 40 30 20 10 0 Hanging Drop Spheroids of HEK293 Cells 160 212 442 1075 Microtissue Diameter (µm) 2015. 22

Yield (ng) RealTime-Glo Reagents Do Not Effect RNA Yield using QuantiFluor RNA System Maxwell + RT-Glo Maxwell - RT-Glo ReliaPrep + RT-Glo ReliaPrep - RT-Glo 1000 100 10 1 160 212 442 1075 Microtissue Diameter (µm) 2013. Proprietary Information. Not for further distribution. 23

RIN values RNA Integrity is Not Affected by Presence of RealTime-Glo Reagent 10 9 8 7 6 5 4 3 2 1 0 RNA Integrity - Bioanalyzer 160 212 442 1075 1075 160 212 442 1075 1075 + RT-Glo -RT-Glo + RT-Glo -RT-Glo Maxwell ReliaPrep Microtissue Diameter (µm) 2015. 24

Advantages of RealTime-Glo Assay Provides kinetic information on viable cell number during the course of experiments that enables on the fly decision making Viable cells remain after applying RealTime-Glo Reagent (i.e. the reagent is not toxic) Remaining viable cells enable a variety of opportunities for multiplexing with other assay chemistries Optional protocols enables reagent to be added when cells are plated, when test compound is added, or at any time point when cell viability measurements are needed. Sensitivity is better than colorimetric or fluorometric viability assays that measure reducing potential of cells. 2015. 26

Real Time Detection of Dead Cells 2015. 27

CellTox Green Cytotoxicity Assay Real Time Method to Detect Dead Cells 2015. 28

DNA Dye Staining to Detect Dead Cells (Overcomes some limitations of short half-life markers) Non-permeable DNA dye Staining of dead cells results in a fluorescent signal that is stable. Viable Cell X Dead Cell 2015. Dye is excluded from live cells DNA dye only stains nucleus of dead cells or debris 29

CellTox Green Dye Measures Accumulation of Dead Cells in Culture HepG2 cells were treated with various doses of Terfenidine. 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. 2015. 30

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 3 days or 15 minutes. Dye is non-toxic for at least 72 hours No effect on IC 50 value of test compounds 2015. 31

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. 2015. 32

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 2015. Record luminescence from apoptotic cells 33

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 2015. 35

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 2015. 36

Questions Welcome If you want a copy of these slides, send email to Terry.Riss@Promega.com 2015. 37