Dead or Alive? New cell-based assays to detect mechanism of toxicity

Transcription

1 Dead or Alive? New cell-based assays to detect mechanism of toxicity Dr Réka Nagy, Promega AG

2 Who we are Using - the bioluminescence of luciferase - Promega has developed a solid technology platform from which hundreds of unique in vitro cell-based, biochemical and metabolic assays have been configured to provide solutions for understanding of cell biology

3 Instrumentation Glo! Fluor Color 3

4 Portfolio of Cell Health Assays Using Plate Readers CytoTox 96 Assay (LDH) Caspase-Glo 3/7 CytoTox-ONE Assay (LDH) Apo-ONE Caspase Assay CellTiter-Fluor Caspase-Glo 8 & 9 CytoTox-Glo (protease) CytoTox-Fluor (protease) MultiTox-Fluor MultiTox-Glo CellTiter-Glo 3D Assay CellTox Green (DNA) RealTime-Glo MT Assay CellTiter 96 Assay (MTT) CellTiter 96 AQ ueous Assay (MTS) CellTiter 96 AQ ueous ONE Soln (MTS) CellTiter-Glo Assay (Luciferin/ Luciferase) CellTiter-Blue GSH-Glo ApoTox-Glo ApoLive-Glo Mitochondrial ToxGlo GSH/GSSG-Glo NAD/NADH-Glo NADP/NADPH-Glo Colorimetric Fluorescence Bioluminescence Fluorescence & Bioluminescence Ultra-Glo Luciferase ROS-Glo H 2 O 2 4

5 Viability CellTiter -Glo, 2.O, 3D Cell Health in Real Time CellTox TM Green RealTime Glo TM (Presented by Thomas) Cell Viability Apoptosis & In Vivo workflow Oxidative Stress & Metabolic Assays ROS-Glo TM H 2 O 2 Assay NAD(P)H Glo TM assay system 5

6 ATP assay for Cell Viability CellTiter-Glo Luminescent Cell Viability Assay Lyses cell membranes to release ATP Inhibits endogenous ATPases Provides luciferin, luciferase and other reagents necessary to measure ATP using a bioluminescent reaction Glow type signal: half-life is 5 hours Extremely sensitive- can detect as few as 10 cells 6

7 Assay Signal (Normalized) CellTiter-Glo 2.0 Assay: Complete Liquid Reagent with Enhanced Shelf Life CellTiter-Glo 2.0 # CellTiter-Glo # CellTiter-Glo stability evaluation performed on reconstituted liquid reagent. # Storage time at 22 o C (weeks) < 20% change in performance 22 o C 4 o C -20 o C CellTiter-Glo 12 hours 3.5 days > 2 yrs CellTiter-Glo days 4 months > 2 yrs #CellTiter-Glo stability evaluation performed on reconstituted liquid reagent. 7

8 What is the CellTiter-Glo 3D Assay? An assay with comparable features to CellTiter-Glo, but with enhanced performance against 3D cell culture models. Key new attributes: liquid format instead of buffer & cake combo enhanced lytic effectiveness (optimized formulation) optimized protocol for 3D model application 8

9 CellTiter-Glo 3D Provides Effective Lysis of Microtissues HCT116 cell spheroids grown to ~350 mm using hanging drop method (InSphero GravityPLUS System) ATPlite 1Step CellTiter-Glo 3D Different lytic effectiveness revealed by membrane-impermeant dye CellTiter-Glo 3D provides near complete lysis of cell spheroid 9

10 Cell Health in RealTime Monitor cells continuously over 72 hours, saving time, cell samples, and costs Option to add reagent at seeding, dosing of compound, or at the end Multiplex with other assays and downstream applications 10

11 CellTox Green Cytotoxicity Assay Real Time Method to Detect Dead Cells 11

12 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. 12

13 CellTox-Green Dye is Not Toxic to Cells ATP assay data showing viability of cells exposed to Bortezomib for 72 hrs O cells exposed to DNA binding dye for 72 hr cells exposed to DNA binding dye for 15 min Cells exposed to dye for 3 days do not show change in viability or responsiveness to toxin. 13

14 CellTox Green Assay: Multiplexing a Fluorescent Assay with Luminescent Assays Glo Reporter Assays NAD(P) / NAD(P)H Will work CellTiter-Glo Cell Viability Assay Glo Reporter Assay Multiplexes include: Nano-Glo One-Glo Bright-Glo Steady-Glo CellTiter-Fluor Viability Assay Caspase-Glo Assays Will work CytoTox- Glo Assay Will work ROS-Glo CellTox Green Cytotoxicity Assay RealTime -Glo Probable BacTiter-Glo Assay P450-Glo Cell-Based Assays GSH-Glo & GSH/GSSG-Glo Assays HDAC-Glo Assay 14

15 Samples with CellTox Green can be Multiplexed with Cell Viability and Apoptosis Assays 24hr GF-AFC 15

16 Viability CellTiter -Glo, 2.O, 3D Cell Health in Real Time CellTox TM Green RealTime Glo TM Cell Viability Apoptosis & In Vivo workflow Oxidative Stress & Metabolic Assays GSH/GSSG-Glo TM Assay ROS-Glo TM H 2 O 2 Assay NAD(P)H Glo TM assay system 16

17 Cellular Markers Indicative of Apoptosis CaspACE Assay Systems (Caspase 3/7) Caspase- Glo 2 Caspase- Glo 3/7 Caspase- Glo 6 Caspase- Glo 8 Caspase- Glo 9 Anti-ACTIVE Caspase-3 pab Anti-PARP p85 Fragment pab DeadEnd Colorimetric TUNEL System DeadEnd Fluorometric TUNEL System 17

18 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 150µM Tamox 24 hr 6 hr Lumi nescence ( Thousands) hr 2 hr 1 hr hr T a m o x i fe n (µ M ) Caspase activity decreases after 24 hours incubation Assay & Drug Devel Tech 2(1): 51,

19 Caspase-Glo 3/7 Assay: Cancer Spheroids and Human Liver Microtissues (30 min shake after reagent addition, then read luminescence) HCT116 spheroids (~340 mm) Human Liver MT (~275 mm) (InSphero hulivmt) (24 hr treatment) (48 hr treatment) Staurosporine Panobinostat 19

20 Viable Cell Multiple multiplex assays: i.e. ApoLive-Glo Multiplex Assay Determine Mechanism of Cell Death measure live cells plus apoptotic cells in the same well Apoptosis Treat cells grown in black or white plates with compound of interest (typically, 0-24 hours) Add Viability Reagent. Apoptosis Mix & incubate 30 minutes 1 3 ProCaspase-3 2 Caspase-3 Live-Cell Protease 2 Necrosis Necrosis 1 Measure Live Cell AFC fluorescence 1 Necrosis Add Caspase-Glo 3/7 Reagent. Mix & incubate 30 minutes ApoLive-Glo Multiplex Assay defines: Apoptotic cell death Necrotic cell death 2 Measure Apoptotic Cell Luminescence Light 20

21 New assay in development Caspase-1: Mediator of Inflammasomes Sollberger et al. (2014) Caspase-1: The inflammasome and beyond Innate Immunity 20:

22 Caspase-Glo 1 Coupled-Enzyme Assay Concept instead of doing Western Blots Z-WEHD- H N aminoluciferin S N N S Caspase-1 COOH Simultaneous reactions Z-WEHD + H 2 N S N COOH N S Ultra-Glo TM Thermostable Luciferase ATP, Mg++ Oxygen Light Light generated is proportional to caspase-1 activity 22

23 In Vivo Imaging Workflow 23

24 Visualizing Liver Apoptosis with VivoGlo Caspase 3/7 Substrate Directly visualizes and quantifies apoptosis non-invasively in mice A rapid and sensitive method Significantly decreases the time for determining preclinical drug efficacy Can be used with any existing cell line or transgenic mouse with firefly luciferase expression Higher signal-to-noise ratio than fluorescent probes 24

25 iability elltiter -Glo, 2O, 3D ell Health in Real Time elltox TM Green ealtime Glo TM Cell Viability poptosis & In Vivo workflow xidative Stress & Metabolic Assays SH/GSSG-Glo TM Assay OS-Glo TM H 2 O 2 Assay AD(P)H Glo TM assay system 25

26 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 ) 26

27 ROS-Glo H 2 O 2 Assay Direct H 2 O 2 detection without using Horseradish Peroxidase (HRP) Mitigates HRP mediated false hits Homogeneous Bioluminescent Assay Add-mix-read No fluorescence interference Cell based assay Detects H 2 O 2 content of culture wells 27

28 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 28

29 Normalized Activity % Amplex Red % Activity vs No Compound Low Interference in LOPAC Chemical Library Screen Amplex Red vs LOPAC Screen ROS Glo vs LOPAC Screen Compound serial # Many compounds in LOPAC interfere with HRP driven Amplex Red assay. 29

30 Oncogenes Drive Cancer by Altering Metabolic Pathways Cancer cells get reprogrammed-uncontrolled cell growth Metabolic pathways get hi-jacked : have to balance energy production with increased needs in biomolecule biosynthesis and ROS production Increase glucose uptake Shift from oxidative phosphorylation to aerobic glycolysis (Warburg effect) Increase lactate production Activates pentose phosphate pathway Increase anabolic reactions 30

31 Principal of Bioluminescent NAD(P)H Detection Technology Assay principal In the presence of NAD(P)H proluciferin is converted to luciferin by reductase Luciferin is used by luciferase to produce light The light is proportional to the amount of NAD(P)H in the sample 31

32 Used the Technology to Develop NAD(P)/NAD(P)H Detection Assays NAD/NADH-Glo and NADP/NADPH-Glo Couples NAD(P)H Detection with Enzyme Cycling Reaction NAD/NADH-Glo Assay detects non-phosphorylated forms in cells NADP/NADPH-Glo Assay detects phosphorylated forms in cells NAD Cycling Product NAD Cycling Substrate NADP Cycling Product NADP Cycling Substrate NAD Cycling Enzyme NADH NAD + NADP Cycling Enzyme NADPH NADP + Reductase Reductase Reductase Substrate Luciferin Reductase Substrate Luciferin 32

33 Three Kits Are Available: NAD(P)H-Glo, NAD/NADH- Glo and NADP/NADPH-Glo The kits can be used for measuring: Total and individual dinucleotides (NAD,NADH, NADP, NADPH) Biochemical enzyme characterization Dinucleotide detection in cells, tissues and other biological samples The NAD/NADH or NADP/NADPH ratio can be determined directly from RLU values The actual amount can be calculated from dinucleotide standard curves 33

34 Features of the Assays: Sensitivity, Specificity and Wide Assay Window NAD(P)H-Glo Detection system NAD/NADH-Glo Detection system NADP/NADPH-Glo Detection system Limit of Detection (LOD) 25nM (625fmol/25ml) 1nM (25fmol/25ml) 1nM (25fmol/25ml) Linearity 25nM 50mM 1 500nM 1 500nM Signal-to-background (S/B max) Cells/well for Total dinucleotides ~400 ~250 ~250 NA , ,000 34

35 Expanding NAD(P)H-Glo Technology to Different Metabolite Detection Metabolite detection assays couple selective metabolite oxidation and NAD(P)H production with bioluminescent NAD(P)H detection Metabolite is used as a substrate by metabolite selective dehydrogenases Simultaneously NAD(P) is converted to NAD(P)H NAD(P)H is detected with reductase/luciferase coupled reactions The light output is proportional to the amount of metabolite present in the sample 35

36 With the aid of auxiliary enzymes nearly every substance of biological interest could be measured with a pyridine nucleotide system by Lowry et al.,

37 Ready-to-use Metabolite Detection Assays Available for Testing: Lactate, Glutamate, Glc-6P, Glucose The assays provide markers for studying Metabolic state of the cells (glycolytic versus Oxphosph) Glycolytic: glucose consumption = lactate secretion Oxphosph: decrease in lactate secretion Mitochondria function Impaired function: increase in glucose consumption and lactate production Glutamine addiction of cancer cells Glucose homeostasis in adipocytes, liver, muscles Note: other metabolite detection assays can be rapidly optimized by using metabolite selective dehydrogenases and coupling it with bioluminescence NAD(P)H detection 37

38 Assay formats: Simple add and read Assays Protocol: Make Metabolite Detection Reagent Add to the samples at 1: 1 ratio Incubate 60 min at room temperature Read luminescence 38

39 Features of Metabolite Detection Assays: Sensitivity, Broad Linearity and Wide Assay Window Sensitivity of the assays allow to measure intracellular metabolite levels directly in 96-,384- well plates Broad linearity of the assays offers more flexibility when comparing the samples at different metabolite levels Wide assay window (S/B max > 100 fold) provides robustness required for high throughput screening approaches Lactate Glucose Glc6P Glutamate Linearity 200mM 22mM 33mM 33mM LOD 90nM 10nM 5nM 15nM S/Bmax

40 Measuring Intracellular Metabolite Levels Measured RLU values and Intracellular metabolite levels calculated from titration curves Note: the media and serum contains significant amounts of different metabolites, therefore for measuring intracellular metabolite levels, the media has to be removed and cells have to be washed with PBS 40

41 Metabolite Detection in Tissues The data are shown with the samples at 1:16 dilution All signals were >8 fold above background Calculated amount of metabolites in liver tissues * RIPA buffer or 50mM Tris, ph7.5 41

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44 Thank you for your attention! Contact details Product Manager Cellular Analysis & Proteomics Réka Nagy, Ph.D. Promega AG Phone: Customer support Promega AG Phone: Technical support Promega AG Phone: