细胞检测方法

Overview of Tools for Cell Biology including Apoptosis Ralph fan Field application specialist Invitrogen corporation Invitrogen Proprietary and Confidential Overview of Tools for Cell Biology Cell Structure • Organelle Probes and Stains • Membrane and Lipid Probes • Cell Tracing Reagents Cell toxicity and apoptosis Invitrogen Proprietary and Confidential Live Cell Tubulin Stain • New TubulinTracker™ Green Better penetration and staining than Oregon Green® paclitaxol Invitrogen Proprietary and Confidential Fixed Tubulin labeling Anti-α-Tubulin mouse IgG2b With Alexa Fluor 350 With Alexa Fluor 568 Zenon Alexa Fluor 488 Invitrogen Proprietary and Confidential F-actin probes (phalloidin) Alexa Fluor® 350 Alexa Fluor® 488 Alexa Fluor® 532 Alexa Fluor® 546 Alexa Fluor® 568 Alexa Fluor® 594 Alexa Fluor® 635 Alexa Fluor® 647 Alexa Fluor® 680 Rhodamine Invitrogen Proprietary and Confidential Mitochondrial Probes • MitoTracker® Green – non-mitochondrial membrane potential dependent – useful for mitochondrial mass • MitoTracker® Red – single wavelength mitochondrial membrane potential dependent – Fixable • JC-1 – ratiometric mitochondrial membrane potential dependent Invitrogen Proprietary and Confidential Mitochondrial Membrane Potential 3T3 mouse fibroblasts labeled with JC-1:Progressive loss of red J-aggregates following H2O2 exposure J C - 1 r e d f l u o r e s c e n c e JC-1 green fluorescence + 50 μM CCCP No Treatment Jurkat Cells + 2 mM JC-1 Invitrogen Proprietary and Confidential Mitochondrial Oxidation MitoSOX™ Red oxidation in live BPAE cells Buffer for 10 min 5 mM MSR 10 mM FeTCPP for 10 min 5 mM MSR Specific for mitochondrial superoxide anion not RNS Invitrogen Proprietary and Confidential MitoSOX™ Red Oxidant Selectivity 0 10000 20000 30000 40000 50000 60000 70000 Su pe rox ide Dy e o nly Su pe rox ide + SO D Su pe rox ide + Fe TC PP HR P + H2 O2 H2 O2 DE AN O Pe rox yn itri te R e l a t i v e F l u o r e s c e n c e U n i t s ( 5 8 0 n m ) Invitrogen Proprietary and Confidential Mitochondrial Transition Pore Assay Schematic of CoQC cell-based assay Invitrogen Proprietary and Confidential Image-iT™ LIVE Mitochondrial Transition Pore Assay - Cobalt quench + Calcium + CsA + Cobalt quench + Calcium -CsA Invitrogen Proprietary and Confidential Live Cell Labeling Lysosomes Live BPAE cells were incubated simultaneously with MitoFluor™ Far Red 680, LysoTracker® Green DND-26, and Hoechst 33342. Invitrogen Proprietary and Confidential SelectFX™ Peroxisome Labeling Kit Fixed and permeabilized BPAE cells labeled with rabbit anti-PMP 70 IgG, Alexa Fluor® 488–labeled goat anti-rabbit IgG, LysoTracker® Red (before fixation; LEFT) or MitoTracker Red® (before fixation; RIGHT), and DAPI Invitrogen Proprietary and Confidential Labeling of Golgi • Golgi – Live • Ceramides – Fixed only • Anti-Golgin 97 antibody • Lectin conjugates Invitrogen Proprietary and Confidential Labeling of ER • Endoplasmic Reticulum – Live • ER-Tracker™ Blue-White (a) • Brefeldin A • New ER-Tracker™ Green (b)/Red (c) (BODIPY® glibenclamide) – Fixed Select FX™ Alexa Fluor® 488 Endoplasmic Reticulum Labeling Kit Invitrogen Proprietary and Confidential Nucleic Acid Counterstains • TOTO® and TO-PRO® Series – Dimer (TOTO®) vs Monomer (TO-PRO®) – TOTO®-3/ TO-PRO®-3 (far red emission) – TO-PRO®-5 (near IR) • SYTOX® Dyes – Blue, green, orange • Traditional Counterstains – DAPI – Propidium Iodide • SelectFX™ Nuclear Labeling Kit with: DAPI, SYTO ® Green, 7-AAD, TO-PRO®-3 iodide Invitrogen Proprietary and Confidential Overview of Tools for Cell Biology Cell Structure • Organelle Probes and Stains • Membrane and Lipid Probes • Cell Tracing Reagents Cell toxicity and apoptosis Invitrogen Proprietary and Confidential Membrane specific Labeling • Dialkylcarbocyanine (DiI, DiO, DiD) ƒ Low fluorescence in water; substantially enhanced fluorescence and photostability upon partitioning into lipid environments ƒ Dye applied locally diffuses laterally to stain entire cell ƒ Useful as long-term tracers • Styryl dyes (FM® 1-43/ FM® 4-64) ƒ Low fluorescence in water; exhibit substantially enhanced fluorescence in the membrane environment ƒ Bind rapidly and reversibly to the plasma membrane ƒ Water-soluble styryl dyes ƒ Useful for studying vesicle recycling FM® 1-43FX fixable form (green/orange emission) New FM® 4-64FX fixable form (far red emission, more hydrophobic) Invitrogen Proprietary and Confidential FM® 4-64FX Live label patterns are comparable. FX version retains label pattern, whereas non-FX version does not. Invitrogen Proprietary and Confidential Overview of Tools for Cell Biology Cell Structure • Organelle Probes and Stains • Membrane and Lipid Probes • Cell Tracing Reagents Cell toxicity and apoptosis Invitrogen Proprietary and Confidential Cell tracers: dextrans and protein tracers Classic examples: fluoro-ruby and fluoro-emerald 10,000 MW dextrans labeled with either rhodamine (ruby) or fluorescein (emerald) •Must be loaded by injection •Long retention time •Some variants are fixable •Group includes protein tracers such as HRP and cholera toxin subunit B Invitrogen Proprietary and Confidential Cell tracers: polar tracers Classic examples: Lucifer Yellow and Sulforhodamine 101 •Similar to dextran tracers, but are low molecular weight •Must be loaded by injection or patch pipette •Some variants are fixable •May cross gap junctions •Also includes labeled hydrazides and biocytin Invitrogen Proprietary and Confidential General Cell Tracker Dyes • Short-term/Non-reactive • CellTrace™ calcein green, AM • CellTrace™ calcein red-orange, AM • Mid-term (72 hours) • CellTracker™ Green CMFDA • CellTracker™ Orange CMTMR • CellTracker™ Red CMTPX • Long-term • CellTracker CM-DiI • CFDA SE (Several kits optimized for different applications) • SNARF-1 carboxylic acid, acetate, SE (S22801) • CellTrace Far Red DDAO-SE Invitrogen Proprietary and Confidential Overview of Tools for Cell Biology Cell Structure Cell toxicity and apoptosis • LIVE/DEAD® cytotoxicity • Apoptosis Invitrogen Proprietary and Confidential Mammalian Viability Assays •Detects live cells using calcein, AM and dead cells using ethidium homodimer-1 to stain nucleus of dead cells •Fluorescence assays are more sensitive than colorimetric assays •Works on live cells; rapid, no wash protocol LIVE/DEAD® Viability/Cytotoxicity Kit Invitrogen Proprietary and Confidential LIVE/DEAD® Fixable Dead cell Stain 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. In both cases, the excess dye is subsequently washed away Microscopy using Fixable Green Dead Cell Stain Flow cytometry analysis using UV laser Unfixed Stain Formaldehyde Fixed Stain Overview of Stain Alternative to EMA: Stain the same before and after fixation Invitrogen Proprietary and Confidential Apoptosis-Vitality 4 hours camptothecin Necrotic (peroxide- treated) APC annexin V SYTOX® Green Dye R e s o r u f i n f l u o r e s c e n c e C12-resazurin resorufin Reductase Vybrant® Apoptosis Assay Kit #10 (V35114) • Allophycocyanin annexin V + C12 resazurin + SYTOX® Green dye • Membrane asymmetry, metabolic activity and membrane integrity • 488 & 633 nm excitation A B C D Jurkat cells treated as labeled: 488 nm & 633 nm excitation Live cells, annexin V-positive, SYTOX® Green-positive Reductase activity / metabolic health Invitrogen Proprietary and Confidential Overview of Tools for Cell Biology Cell Imaging Cell toxicity and apoptosis • LIVE/DEAD® cytotoxicity • Apoptosis Invitrogen Proprietary and Confidential Vybrant® FAM Caspase Assay Kits Mitochondria dATP Calpain Activation Ca2+ Chemical or γ Irradiation Energy Free Radicals NAD PARPO2 O2Bcl-2 Cyto c Cyto c Cyto c APAF-1 APAF-1 pCasp-9 pCasp-9 Caspase-9 Effector Caspase Ca2+ ↓ΔΨm AIF EndoG DNA Fragmentation DNA Damage Apoptosis Necrosis Caspase-3 Caspase-6 Caspase-7 Apoptosome • Vybrant® FAM Caspase-3 and –7 Assay Kit (V35118) • Vybrant® FAM Caspase-8 Assay Kit (V35119) • Vybrant® FAM Polycaspases Assay Kit (V35117) • Z-DEVD (E13183, E13184, A22120, A22121) • Z-IETD (A22127, A22125, A22128) Invitrogen Proprietary and Confidential FLICA™ Function Detection of active caspases in apoptotic cells Invitrogen Proprietary and Confidential Caspase Assay Kits for Imaging I35101 Image-iT™ LIVE Red Poly Caspases Detection Kit I35102 Image-iT™ LIVE Red Caspase-3 and -7 Detection Kit I35104 Image-iT™ LIVE Green Poly Caspases Detection Kit I35105 Image-iT™ LIVE Green Caspase-8 Detection Kit I35106 Image-iT™ LIVE Green Caspase-3 and -7 Detection Kit Healthy Cells Apoptotic Cells Red Caspase 3/Sytox® Green Dye/Hoechst Apoptotic Cells Green Caspase 3/ PI/Hoechst Invitrogen Proprietary and Confidential Detecting Mitochondrial Changes Mitochondria dATP Calpain Activation Ca2+ Chemical or γ Irradiation Energy Free Radicals NAD PARPO2 O2Bcl-2 Cyto c Cyto c Cyto c APAF-1 APAF-1 pCasp-9 pCasp-9 Caspase-9 Effector Caspase Ca2+ ↓ΔΨm AIF EndoG DNA Fragmentation DNA Damage Apoptosis Necrosis Caspase-3 Caspase-6 Caspase-7 Apoptosome • JC-1 • MitoTracker® Red CMXRos • Vybrant® Apoptosis Assay Kit #11 (V35116) • MitoProbe™ JC-1 Assay Kit (M34152) • MitoProbe™ DiOC2(3) Assay Kit (M34150) • MitoProbe™ DiIC1(5) Assay Kit (M34151) • MitoProbe™ Transition Pore Assay Kit (M34153) Invitrogen Proprietary and Confidential Apoptosis-Mitochondria MitoProbe™ DiOC2(3) Assay Kit (M34150) • Mitochondrial membrane potential • 488 nm excitation – uses all emissions • Easier to use than JC-1; better population resolution • Can be used with APC-Annexin V conjugates for excellent multiparameter study of Apoptosis and Vitaliy DiOC2(3) on HeLa cells Decrease of red and green DiOC2(3) signals in apoptotic cells +CCCP Easy reagent – clear result Invitrogen Proprietary and Confidential Apoptosis- Mitochondria E v e n t s DiIC1(5) fluorescence +50 μM CCCP Untreated Jurkat cells stained with 50 nM DiIC1(5): 633 nm Excitation MitoProbe™ DiIC1(5) Assay Kit (M34151) • Mitochondrial membrane potential • 633 nm excitation – allows use of 488 nm emissions for other reagents • Easier to use than JC-1; better population resolution Decrease of far red DiIC1(5) fluorescence DiIC1(5) on MRC5 cells Detection of mitochondrial changes with a 633nm laser Invitrogen Proprietary and Confidential Cell Function Probes-Apoptosis 4 Hour Staurosporine TreatmentUntreated SelectFX™ Alexa Fluor® 488 Cytochrome c Labeling Kit Invitrogen Proprietary and Confidential Reactive Oxygen Species Detection Product Peak Excitation Peak Emission ROS species cis-parinaric acid 304 nm 416 nm ROO., H2DCFDA, Carboxy- or CM- H2DCFDA 505 nm 530 nm H2O2, HO ., ROO.,ONOO- Dihydroethidium 518 nm 605 nm .O2- Dihydrorhodamine 123 507 nm 530 nm H2O2, HOCl, ONOO- Dihydrorhodamine 6G 528 nm 550 nm H2O2,ONOO - Hydroxyphenyl Fluorescein (HPF) 490 nm 515 nm HO., ONOO- Aminophenyl Fluorescein (APF) 490 nm 515 nm HO., HOCl, ONOO- RedoxSensor™ Red 540 nm 600 nm H2O2, ROO ., .O2- Singlet Oxygen Sensor Green Before 372, 393 After 504 nm 395, 416 nm 525 nm O2 Invitrogen Proprietary and Confidential Loss of Membrane Asymmetry Untreated cells Jurkat cells stained with Vybrant® Apoptosis Assay Kit #2 (V13241) Induced with camptothecin Apoptotic Dead Viable Apoptotic Dead Viable Annexin V Conjugates (AF350, AF488, AF568, RPE, AF594, AF647, APC) Invitrogen Proprietary and Confidential Plasma Membrane Permeability Assays Apoptosis induced in Jurkat cells with 10 µM camptothecin Vybrant® Apoptosis Assay Kit #4 ƒ Apoptotic cell nuclei were labeled with YO-PRO®-1 dye (green) ƒ Necrotic cells were detected with propidium iodide (red) Invitrogen Proprietary and Confidential Apoptosis- Monomeric Cyanine Dyes YO-PRO-1 dye fluorescence 488 nm excitation P I f l u o r e s c e n c e 4 8 8 n m e x c i t a t i o n Live Apoptotic Dead TO-PRO-3 dye fluorescence 633 nm excitation S Y T O X G r e e n d y e f l u o r e s c e n c e 4 8 8 n m e x c i t a t i o n PO-PRO-1 dye fluorescence 405 nm excitation P I f l u o r e s c e n c e 4 8 8 n m e x c i t a t i o n Chemically-defined dye combinations can be selected that correlate with annexin V/impermeant dye discrimination of healthy, apoptotic, and dead cells using virtually any visible excitation wavelength. YO-PRO-1 dye fluorescence 488 nm excitation A n n e x i n V A P C f l u o r e s c e n c e 6 3 3 n m e x c i t a t i o n TO-PRO-3 dye fluorescence 633 nm excitation A n n e x i n V R P E f l u o r e s c e n c e 4 8 8 n m e x c i t a t i o n PO-PRO-1 dye fluorescence 405 nm excitation A n n e x i n V A P C f l u o r e s c e n c e 6 3 3 n m e x c i t a t i o n Advantages •Does not require any special buffers •Does not rely upon conjugate chemistry •Can be used on trypsinized cells Invitrogen Proprietary and Confidential Chromatin Condensation / DNA Fragmentation Vybrant® Apoptosis Assay Kit #5 (V13244) Hoechst 33342 / propidium iodide APO-BrdU TUNEL Assay Kit (A23210) Alexa Fluor® 488 anti-BrdU / propidium iodide Invitrogen Proprietary and Confidential Timeframes within Camptothecin / Jurkat Model* Relative TimeFunction Reagent PS Translocation Annexin Membrane Integrity PI, SYTOX® Green dye Mitochondria Activity Changes Membrane Potential Transition Pore MitoTracker® Red dye DiOC2(3), DiIC1(5), JC-1 MTP assay Caspase Activity Caspase substrates Metabolic Activity C12 resazurin DNA Condensation Hoechst DNA Fragmentation TUNEL ASSUMED Increase Decrease Partial Change * Jurkat cells induced with 10 µM camptothecin Putting it into a framework Invitrogen Proprietary and Confidential Selection Guide for Vybrant® Apoptosis Kits Kit # Cat # Viable Cell Stain Apoptotic Cell Stain Dead Cell Stain Excitation Kit #1 V13240 None AF488-Annexin V SYTOX® Green 488nm Kit #2 V13241 None AF488-Annexin V PI (Red) 488nm Kit #3 V13242 None FITC-Annexin V PI (Red) 488nm Kit #4 V13243 None YO-PRO® -1 (Green) YO-PRO® -1 PI (Red) 488nm Kit #5 V13244 Hoechst 33342 Dim Hoechst 33342 (Blue)-Bright PI (Red) UV & 488nm Kit #7 V23201 Hoechst 33342 Dim YO-PRO® -1 Hoechst 3342 YO-PRO® -1 PI (Red) UV & 488nm Kit #8 V35112 None R-Phycoerythrin- Annexin V (Orange) SYTOX® Green 488nm Kit #9 V35113 None Allophycocyanin- Annexin V (Far Red) SYTOX® Green 488nm & 633nm Kit #10 V35114 C12-resauzurin (Red) Allophycocyanin- Annexin V (Far Red) SYTOX® Green 488nm & 633nm Kit #11 V35116 MitoTracker® Red AF488-Annexin V None 488nm Kit #12 V35121 DyeCycle Violet DyeCycle Violet 7-AAD 405nm & 488 or 532 Kit #13 V35123 None PO-PRO-1 7-AAD 405nm & 488 or 532 Kit #14 V35124 None Pacific Blue Annexin V 7-AAD 405nm & 488 or 532 Invitrogen Proprietary and Confidential Overview of Tools for Cell Biology Thank You!