Investigating Apoptosis. For more information:
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1 Investigating Apoptosis For more information:
2 Flow Cytometry: It s in our DNA October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 2
3 Two Facets of Viability Viability detection Easy additions to panels Traditional reagents (DNA dyes) Fixable assays (amine-reactive dyes) Apoptosis detection Value of multiple parameters Mitochondrial changes Membrane events Caspases Nuclear changes October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 3
4 Why Use a Viability Indicator? Perfetto et al. (2006) J Immunol Methods 313:199 October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 4
5 Range of Viability Dyes Name Excitation Maximum Emission Maximum Excitation Laser Suggested Filter SYTOX Blue stain 445 nm 470 nm 405 nm 450/50 Traditional Dyes (DNA Dyes) SYTOX Green stain Propidium iodide 7-aminoactinomycin D SYTOX AADvanced stain 504 nm 535 nm 546 nm 546 nm 523 nm 617 nm 647 nm 647 nm 488 nm 488 nm 488 nm 488 nm 530/30 610/20 650BP or 670BP 650BP or 670BP SYTOX Red stain 640 nm 650 nm 633 nm 660/20 LIVE/DEAD Fixable Blue Dead Cell Stain 350 nm 450 nm UV 450/50 LIVE/DEAD Fixable Violet Dead Cell Stain 416 nm 451 nm 405 nm 450/50 Fixable Dyes (Amine-reactive) LIVE/DEAD Fixable Aqua Dead Cell Stain LIVE/DEAD Fixable Green Dead Cell Stain LIVE/DEAD Fixable Red Dead Cell Stain 367 nm 495 nm 595 nm 526 nm 520 nm 615 nm 405 nm 488 nm 488 nm 525/50 530/30 610BP or 620BP LIVE/DEAD Fixable Far Red Dead Cell Stain 650 nm 665 nm 633 nm 660/20 LIVE/DEAD Fixable Near IR Dead Cell Stain 750 nm 775 nm 633 nm 780/60 October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 5
6 Traditional Dyes: Impermeant Nucleic Acid Stains October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 6
7 SYTOX Red Dump Channel October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 7
8 Native Cell Proliferation: Live Cell Gating October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 8
9 Viability Measure in Apoptotic Cells October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 9 Jurkat cells + camptothecin
10 LIVE/DEAD Fixable Dead Cell Stains October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 10
11 LIVE/DEAD Fixable Dead Cell Stains October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 11
12 LIVE/DEAD Fixable Dead Cell Stains October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 12
13 Breakthrough Cell Proliferation Assay Click-iT EdU Cell Proliferation Assay for Flow Cytometry Click-iT Alexa Fluor 488 azide Cell Cycle 633-red stain October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 13
14 EdU Labeling October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 14
15 Polyploidy + Dead Cell Discrimination October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 15
16 Apoptosis Programmed Cell Death Ca 2+ Chemical or γ Irradiation Ca 2+ datp Calpain Activation Bcl-2 O 2 O 2 PARP NAD Apoptosome pcasp-9 APAF-1 APAF-1 pcasp-9 Cyto c Cyto c Effector Caspase Cyto c Caspase-3 Caspase-6 Caspase-7 ΔΨ m EndoG Mitochondria Energy Free Radicals Caspase-9 AIF DNA Fragmentation DNA Damage Apoptosis Necrosis October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 16
17 Timeframes within Camptothecin / Jurkat Model* October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 17
18 MitoProbe Reagents October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 18
19 Carbocyanine Dyes: MitoProbe JC-1 dye October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 19
20 Carbocyanine Dyes: MitoProbe DiIC1(5) dye October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 20
21 MitoTracker Red CMXRos Dye October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 21
22 Mitochondrial Permeability Transition Pore October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 22
23 MitoProbe Transition Pore Assay October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 23
24 MitoProbe Transition Pore Assay: BPAE Cells October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 24
25 Loss of Membrane Asymmetry / Loss of Integrity October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 25
26 Loss of Membrane Asymmetry: Annexin V October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 26
27 Vybrant FAM Caspase Assay Kits October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 27
28 Vybrant FAM Caspase Assay Kits: FLICA Function October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 28
29 Vybrant FAM Caspase Assay Kits Propidium Iodine Fluorescence FAM Fluorescence October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 29
30 Vybrant FAM Caspase Assay Kits Propidium Iodine Fluorescence Dead Cells PI Positive Live Cells FAM Negative Apoptotic Cells FAM Positive FAM Fluorescence October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 30
31 Detecting Nuclear Changes October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 31
32 Nuclear Condensation October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 32
33 Detection of Sub-G1 Population & DNA Fragmentation October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 33
34 TUNEL (TdT mediated dutp Nick End Labeling) October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 34
35 Click-iT TUNEL Alexa Fluor Imaging Assay October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 35
36 Jurkat Cells: Reverse Timecourse - 4 hours - 3 hours 10 μm Camptothecin - 2 hours - 1 hours - 30 minutes Harvest & stain at time 0 October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 36
37 Jurkat Cells: Reverse Timecourse - 4 hours - 3 hours 10 μm Camptothecin - 2 hours - 1 hours - 30 minutes Harvest & stain at time 0 October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 37
38 Jurkat Cells: Time After Camptothecin Induction October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 38
39 Jurkat Cells: Time After Camptothecin Induction DiIC 1 (5) (661BP) JC-1 red (670LP) 488 nm & 633 nm Excitation Annexin V-APC (661BP) 488 nm & 633 nm Excitation Annexin V-Alexa Fluor 488 dye (530BP) October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 39
40 Jurkat Cells: Appearance of Sub-G 1 Peak October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 40
41 Timeframes within Camptothecin / Jurkat Model* October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 41
42 Reagent Use on Cytometers: FACS Calibur System Flow Instrumentation Guide (PDF, October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 42
43 Reagent Use on Cytometers: FACS Calibur System Flow Instrumentation Guide (PDF, Proliferation Viability Calcium Apoptosis / Viability Proliferation Transfection Apoptosis Viability Proliferation Apoptosis Viability Apoptosis / Viability Viability Calcium Proliferation Apoptosis ph ph Viability Viability ph Apoptosis Viability Proliferation Viability Apoptosis / Viability Viability Apoptosis Proliferation October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 43
44 Acknowledgments Resources Invitrogen Gayle Buller Jolene Bradford Diane Gray Stephen Yue JiaXiang Liu Timothy Huang Patrick Pinson Yu-Zhong Zhang Pam Whitney Scott Clarke Brian Agnew Dani Hill Yih-Tai Chen Iain Johnson Suzanne Buck Kyle Gee Kathy Free Mike Olszowy Online Fluorescence & Flow Cytometry Tutorials NIH William Telford Mario Roederer Stephen Perfetto Practical Flow Cytometry Howard Shapiro iprotocols BD FACS Calibur Flow Instrumentation Guide Spectraviewer APO-BrdU is a registered trademark of Phoenix Flow Systems Cy is a registered trademark of GE Healthcare Posters from recent conferences October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 44
45 THANK YOU! Questions? October 2, Invitrogen Corporation. All Rights Reserved. Proprietary and Confidential. 45
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