Assessing Cell Health and Viability Ian Clements. Molecular Probes Life Technologies Inc.

Transcription

1 Assessing Cell Health and Viability Ian Clements Molecular Probes Life Technologies Inc.

2 Cell Health, Viability and Vitality Cell Health by Cell Counting Viability Determination Live vs Dead Vitality Determination How healthy/sick cells are? Special Case: Apoptosis July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 2

3 Cell Health, Viability and Vitality Cell Health by Cell Counting Montoring cell health by changes in cell number within population. Viability Determination Live vs Dead Vitality Determination How healthy/sick cells are? Special Case: Apoptosis July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 3

4 Approaches to Cell Number Determination Cell Number by Metabolic Indicators Vybrant Cell Metabolism Assay Kit LIVE/DEAD Calcein AM Vitality Kits Luminescent: ATP Determination Kit Colorimetric: Alamar Blue, MTT Cell Number by DNA Content CyQuant, CyQuant NF Cell Division by DNA synthesis BrdU, antibrdu Click-iT EDU Cell Division by Tracer Dye Analysis CFSE (CellTrace kits and reagent) Vybrant DiI Cell Division by Cell Cycle Analysis DyeCycle Dyes Other Solutions Counting Beads. July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 4

5 Reagent summary for cytotoxicity or proliferation analysis alamarblue CyQuant Assays Click-iT EdU Kits Product Answers the question: What is the metabolic health of the cells? Have my cells multiplied? How many cells are present? Which cells are actively proliferating? Primarily used for: Cell viability and cytotoxicity, & indirect proliferation measurement Cell counting via DNA content to assess cytotoxic or proliferative effects independent of cellular metabolism Direct indicator of cell proliferation Why choose: Easy, HTP, homogeneous assay Economical Non toxic to cells No wash assay Highly accurate, sensitive, and linear Workflow choices Extremely rapid Easy Edu replaces difficult BrdU assays Samples remain intact Imaging, HCS, flow and microplate formats How it works: Metabolic assay for cellular reducing environment where readout is proportional to number of living cells Quantitates relative number of cells in a population based on total DNA content by measuring DNA binding dye Measures rate of new DNA synthesis by EdU nucleoside analog incorporation into DNA with detection by copper-catalyzed click reaction July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 5

6 Countess is a complete counting platform C10227 Countess automated cell counter) Includes Countess instrument, box of 50 slides and trypan blue C10228 Countess cell counting chamber slides, 50 Includes trypan blue *C10288 Countess field test kit (for internal sales only) Includes box of 50 slides, trypan blue, beads and instructions July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 6

7 Countess Software Look at cell count July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 7

8 Countess data has a wider range than hemocytometer 1.00E+07 Countess automated cell counter Hemocytometer Measured concentration 1.00E+06 Countess Range 1x10 4 1x10 7 cells/ml Highest accuracy: 1x10 5 4x10 6 cells/ml Hemocytometer range 2.5x x10 6 cells/ml 1.00E E E E+07 Cell suspension dilution July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 8

9 Cell Health, Viability and Vitality Cell Health by Cell Counting Viability Determination Assessing overall number of living vs dead cells in the population. Vitality Determination Assessing the health of cells in the population. Ideally combined with viability determination. Special Case: Apoptosis July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 9

10 Viability & Vitality Viability: Cell membrane forms intact barrier Vitality: Cell mediates active processes Cytosol Nucleus Cytosol Nucleus + Propidium iodide + Carboxyfluorescein Nonviable (Dead) Nonvital (Dead) Viable (Live) Vital (Live) July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 10

11 Viability & Vitality: Individual Reagent Examples Viability Vitality Permeant DNA Dyes (Live & Dead) SYTO 9 dye SYTO 13 dye SYTO 16 dye SYTO 25 dye SYTO 59 dye Impermeant DNA Dyes (Dead) SYTOX Green Propidium iodide (PI) 7-AAD TO-PRO -3 dye Ethidium homodimers Cell Function Markers Esterase CFDAs Calcein AM Oxidation Dihydrorhodamine 123 C 12 -resazurin (Vybrant Cell Metabolic Assay) Alamar Blue July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 11

12 Impermeant Nucleic Acid Dyes Cell-Impermeant Nucleic Acid Dyes: Penetrate compromised cell membranes stain nucleic acids in dead cells do not cross the membranes of live cells Examples include: Propidium Iodide dsdna/rna 7-AAD SYTOX Green stain SYTOX Red stain dsdna/rna DNA/RNA DNA/RNA July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 12

13 Impermeant Nucleic Acid Stains SYTOX Green 7-Aminoactinomycin D Propidium Iodide SYTOX Green Fluorescence 488 excitation SYTOX Red 7-AAD Fluorescence 488 excitation Propidium Iodide Fluorescence 488 excitation SYTOX Red Fluorescence 633 excitation July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 13

14 Fixable Dead Cell Stains LIVE/DEAD Reduced Biohazard Cell Viability Kit #2 Principal LIVE/DEAD Fixable Green Dead Cell Stain Kit (L23101) Healthy cells Aged culture Live cells (left) react with the kit s fluorescent reactive dye only on their surface to yield weakly fluorescent cells. Cells with compromised membranes (right) react with the dye throughout their volume, yielding brightly stained cells. Heat-killed cells In both cases, the excess reactive dye is subsequently washed away. July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 14

15 Summary of Viability Dyes Name Excitation Maximum Emission Maximum S34857 SYTOX Blue stain 445 nm 470 nm Traditional Dyes (DNA Dyes) S7020 SYTOX Green stain P1304 Propidium iodide A aminoactinomycin D S10349 SYTOX AADvanced stain 504 nm 535 nm 546 nm 546 nm 523 nm 617 nm 647 nm 647 nm S34859 SYTOX Red stain 640 nm 650 nm L23105 LIVE/DEAD Fixable Blue Dead Cell Stain 350 nm 450 nm Fixable Dyes (Amine-reactive) L34955 LIVE/DEAD Fixable Violet Dead Cell Stain L34957 LIVE/DEAD Fixable Aqua Dead Cell Stain L23101 LIVE/DEAD Fixable Green Dead Cell Stain L23102 LIVE/DEAD Fixable Red Dead Cell Stain L10120 LIVE/DEAD Fixable Far Red Dead Cell Stain 416 nm 367 nm 495 nm 595 nm 650 nm 451 nm 526 nm 520 nm 615 nm 665 nm L10119 LIVE/DEAD Fixable Near IR Dead Cell Stain 750 nm 775 nm July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 15

16 Vitality by Enzyme / Metabolic Function Cytosol Live Cell Nucleus Calcein AM Ethidium Homodimer-1 Dead Cell AM cleavage July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 16

17 LIVE/DEAD Viability / Cytotoxicity Kit (L3224) Calcein AM and Ethidium Homodimer-1 Rapid assay Detects live and dead cells simultaneously The top selling viability assay kit July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 17

18 LIVE/DEAD Cell Vitality Assay Kit (L34951) % fluorescence remaining in cells C 12 resazurin + SYTOX Green dye Metabolic activity and viability 488 excitation C 12 resazurin conversion to resorufin C 12 resazurin alamarblue Jurkat cells induced with 10 μm camptothecin for 4 hours, then stained with the LIVE/DEAD Cell Vitality Assay Kit SYTOX Green dye fluorescence Time (minutes) C 12 resazurin fluorescence July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 18

19 Viability & Vitality: LIVE/DEAD Kits Sku # Kit Name Live Stain Dead Stain Fix? L3224 LIVE/DEAD Viability/ Cytotoxicity Kit Calcein AM Ethidium Homodimer-1 No L7010 LIVE/DEAD Cell- Mediated/Cytotoxicity Kit DiOC 18 (3) Propidium Iodide No L7013 LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 SYTO 10 dye Reactive Red Yes L23101 LIVE/DEAD Reduced Biohazard Cell Viability Kit #2 Blue Dye dim Blue Dye bright Yes L23102 LIVE/DEAD Reduced Biohazard Cell Viability Kit #3 Red Dye dim Red Dye bright Yes L23105 LIVE/DEAD Reduced Biohazard Cell Viability Kit #4 Green Dye dim Green Dye bright Yes L7011 LIVE/DEAD Sperm Viability Kit SYBR 14 dye Propidium Iodide No July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 19

20 Cell Health in Imaging and HCS Flow cytometry and plate readers are the primary platforms for health and viability assays Automated Imaging or HCS adds the dimension of spatial information to the analysis. July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 20

21 The Mitochondrial respiratory chain: A sensitive indicator of pre-lethal cytotoxicity Intermembrane space ROS ROS Matrix July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 21

22 Cell Vitality using Mitochondrial Stains. Total dye concentration in all three images is the same + 1 µm FCCP Self-quenching of rhodamine 123 (R302) in rat cortical astrocytes July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 22

23 MitoProbe Transition Pore Assay Kit (M34153) Detects early mitochondrial membrane event Direct detection of the mitochondrial permeability transition pore (MPTP) 488 nm excitation & green emission; use of orange/red channels for other reagents D E A B C July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 23

24 Image-iT LIVE Mitochondrial Transition Pore Assay Kit (I35103) BPAE cells A labeled with calcein AM, MitoTracker Red dye, and Hoechst D C No Treatment With Ionomycin July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 24

25 MitoProbe JC-1 Assay Kit (M34152) Mitochondrial membrane potential New packaging of classic dye with robust protocol 488 nm excitation uses all emission Widely Used July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 25

26 HCS Mitochondrial Health Kit 4.4 μm 13 μm 40 μm 120 μm Hoechst (Nuclear Morphology) Image-IT DEAD Green TM Viability stain (Cell Membrane Permeability) MitoHealth stain (Mitochondrial Membrane Potential) July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 26

27 Detection of pre-lethal mitotoxicity and lethal cytotoxicity in HeLa cells Fluorescence (a.u.) dmso Mitochondrial Membrane Potential val 30 val 120 CCCP 20 CCCP 80 Trog 40 Trog 105 Mitochondrial Membrane Potential HeLa - Valinomycin EC 50 = 5.5 nm Log Valinomycin (μm) Fluorescence (a.u.) dmso val 30 Cell Membrane Permeability val 120 CCCP 20 CCCP 80 Trog 40 Trog 105 Cell Membrane Permeability HeLa - Valinomycin 1000 EC 50 =25.5 μm Log Valinomycin (μm) July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 27

28 Live Cell Reactive Oxygen Species Detection PBS 0.03% H 2 O 2 Image-iT LIVE Green Reactive Oxygen Species Detection Kit (I36007) Jurkat cells incubated with 100 nm CM-H 2 DCFDA (C6827) 5-(and-6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate, acetyl ester 488 nm excitation 525 nm emission Untreated TBHP Treated July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 28

29 Reactive Oxygen Species Detection Reactive Oxygen Species Hydrogen peroxide Hydroxyl radical Hypochlorous acid Nitric oxide Peroxyl radical, including both alkylperoxyl and hydroperoxyl radicals (wherein R = H) Peroxynitrite anion Superoxide anion Structure H 2 O 2 HO HOCl NO ROO ONOO O 2 Detection Reagents Carboxy-H 2 DCFDA (C400) CM-H 2 DCFDA (C6827) Dihydrocalcein AM (D23805) Dihydrorhodamine 123 (D632, D23806) H 2 DCFDA (D399) CM-H 2 DCFDA (C6827) Dihydrorhodamine 123 (D632, D23806) DAF-FM diacetate (D23842, D23844) H 2 DCFDA (D399) Carboxy-H 2 DCFDA (C400) CM-H 2 DCFDA (C6827) H 2 DCFDA (D399) Carboxy-H 2 DCFDA (C400) CM-H 2 DCFDA (C6827) Dihydrorhodamine 123 (D632, D23806) Dihydroethidium (D1168, D11347, D23107) July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 29

30 HCS DNA Damage Kit for genotoxicity and cytotoxicity DMSO 30 µm 120 µm DNA damage (ph2ax antibody/alexa Fluor 555 secondary) Cell Membrane Permeability (Image-iT DEAD Green viability stain) Nuclear morphology (Hoechst 33342) A549 Cells 24h Valinomycin July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 30

31 HCS LipidTOX phospholipidosis detection reagents Perturbation of lipid metabolism and the resulting lipid accumulation is a common side-effect of many pharmaceuticals Seed cells Drug & Labeled Phospholipid coincubation HepG2 cells co-incubated with HCS LipidTOX Green phospholipidosis detection reagent and +/- 10 μm chloroquine for 48 hours before fixation Formaldehyde fixation Imaging & Analysis July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 31

32 HCS LipidTOX neutral lipid stains for steatosis Mechanism of staining Specific recognition of neutral lipids Convenience Ready to use DMSO solution and no wash steps required Flexibility Green, red, and infrared emission choices with tight emission spectra for multiplexing HepG2 cells incubated with +/- 10 μm CsA for 48 hours before fixation and labeling with HCS LipidTOX Green neutral lipid detection reagent Seed cells Drug Incubation Formaldehyde fixation Neutral lipid staining Imaging & Analysis July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 32

33 LipidTOX neutral lipid stains for adipogenesis applications Adipocytes differentiated from human mesenchymal stem cells, fixed, and labeled with LipidTOX Green or Deep Red neutral lipid stain and Hoechst Adipocytes differentiated from 3T3 L1 mouse fibroblasts, fixed, and labeled with LipidTOX Green neutral lipid stain, anti-fabp4 (red), and Hoechst July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 33

34 phrodo BioParticles for Phagocytosis phrodo : ph-sensitive fluorescence ph 10 ph 8 ph 7 ph 6 ph 5 ph 4 Fl (rfu) λ (nm) P35261 phrodo E. coli BioParticles A10010 phrodo S. aureus BioParticles July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 34

35 Endocytosis: phrodo conjugates phrodo -Conjugate Hoechst Mulitplexing with phrodo dye (P36600) OVERLAY Endosome-GFP vehicle 40μM Dynasore phrodo AFL-647-TfN July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 35

36 TUNEL (TdT mediated dutp Nick End Labeling) July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 36

37 TUNEL Detection of apoptotic HeLa cells Click-iT TUNEL Alexa Fluor Imaging Kits C10245 Alexa Fluor 488 Kit C10246 Alexa Fluor 594 Kit C10247 Alexa Fluor 647 Kit 0.5 um staurosporine Click-iT TUNEL Alexa Fluor 594 Anti-cleaved caspase 3 /Alexa Fluor 488 GAR Hoechst um staurosporine Click-iT TUNEL Alexa Fluor 647 Anti-cleaved caspase 3 /Alexa Fluor 488 GAR Anti-tubulin/ Alexa Fluor 555 GAR Hoechst July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 37

38 Cell Health, Viability and Vitality Cell Health by Cell Counting Viability Determination Live vs Dead Vitality Determination How healthy/sick cells are? Special Case: Apoptosis July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 38

39 Apoptosis Detection Function Reagent Relative Time Mitochondria Activity Changes Membrane Potential Transition Pore PS Translocation Caspase Activity Metabolic Activity DNA Condensation Membrane Integrity MitoTracker Red dye DiOC 2 (3), DiIC 1 (5), JC-1 MTP assay Annexin Caspase substrates C 12 resazurin Hoechst PI, SYTOX Green dye DNA Fragmentation TUNEL ASSUMED Increase Decrease Partial Change * Jurkat cells induced with 10 µm camptothecin July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 39

40 Detectable Cellular Changes During Apoptosis? Membrane structure annexin V, YO-PRO -1 dye (Vybrant apoptosis kits) Mitochondrial membrane function MitoTracker Red dye MitoProbe kits (JC-1, DiOC 2 (3), DiIC 1 (5)) Cytochrome c translocation MitoProbe Transition Pore Assay Kit Proteolysis Vybrant FAM caspase kits Cellular metabolism C 12 -resazurin (Vybrant Apoptosis Kit #10, LIVE/DEAD Cell Vitality Kit) Membrane integrity: propidium iodide, SYTOX Green dye DNA condensation & fragmentation: Hoechst dyes, TUNEL July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 40

41 Selection Guide for Vybrant Apoptosis Assay Kits Kit # Cat # Viable Cell Stain Apoptotic Cell Stain Dead Cell Stain Kit #1 V13240 None Alexa Fluor 488 dye -Annexin V SYTOX Green Kit #2 V13241 None Alexa Fluor 488 dye - Annexin V PI (Red) Kit #3 V13242 None FITC-Annexin V PI (Red) Kit #4 V13243 None YO-PRO -1 dye (Green) YO-PRO -1 dye PI (Red) Kit #5 V13244 Hoechst dim Hoechst (Blue)-Bright PI (Red) Kit #7 V23201 Hoechst dim YO-PRO -1 dye Hoechst 3342 YO-PRO -1 dye PI (Red) Kit #8 V35112 None R-Phycoerythrin-Annexin V (Orange) SYTOX Green dye Kit #9 V35113 None Allophycocyanin-Annexin V (Far Red) SYTOX Green dye Kit #10 V35114 C 12 -resauzurin (Red) Allophycocyanin-Annexin V (Far Red) SYTOX Green dye Kit #11 V35116 MitoTracker Red dye Alexa Fluor 488 dye - Annexin V None July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 41

42 Vybrant Apoptosis Assay Kit #10 (V35114) Allophycocyanin annexin V + C 12 resazurin + SYTOX Green dye Membrane asymmetry, metabolic activity and membrane integrity 488 & 633 nm excitation Jurkat cells treated as labeled: 488 nm & 633 nm excitation Live cells, annexin V-positive, SYTOX Green-positive A B C 12 -resazurin resorufin Reductase 4 hours camptothecin Necrotic (peroxidetreated) Resorufin fluorescence C D APC annexin V SYTOX Green Dye July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 42

43 THANK YOU! Questions? July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 43

44 Stains for cell demarcation and image segmentation Staining of Entire (Fixed) Cells H32720 HCS CellMask Blue stain H32714 HCS CellMask Green stain (new) H32713 HCS CellMask Orange stain (new) H32712 HCS CellMask Red stain (new) H32721 HCS CellMask Deep Red stain Prominent Nuclear Staining in (Live or Fixed) Cells H10325 HCS NuclearMask Blue stain (new) H10326 HCS NuclearMask Red stain ( new ) H10294 HCS NuclearMask Deep Red stain Fixable Plasma Membrane Staining in (Live) Cells C10045 CellMask Orange Plasma Membrane stain C10046 CellMask Deep Red Plasma Membrane stain July 7, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 44