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Fluorescent Solutions in Cell Biology Recent Developments in Cellular Imaging, Apoptosis, Viability and Cell Proliferation Dana Brozban Technical Sales.

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Presentation on theme: "Fluorescent Solutions in Cell Biology Recent Developments in Cellular Imaging, Apoptosis, Viability and Cell Proliferation Dana Brozban Technical Sales."— Presentation transcript:

1 Fluorescent Solutions in Cell Biology Recent Developments in Cellular Imaging, Apoptosis, Viability and Cell Proliferation Dana Brozban Technical Sales Specialist Imaging and Microscopy

2 2 This Presentation Focus on Key Molecular Probes Products and Applications “The Illuminated Cell” Apoptosis Assays, Viability and Cell Proliferation

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4 Nucleus T3605 TO-PRO®-3 iodide S34857 SYTOX® Blue S7020 SYTOX® Green S11368SYTOX® Orange D1306DAPI H3570 Hoechst SYTO® 9 dye SYTO® 13 dye SYTO® 16 dye SYTO® 25 dye SYTO® 59 dye

5 Nucleoli S32703SYTO® RNASelect™ green fluorescent cell stain

6 Endoplasmic Reticulum E12353ER-Tracker™ Blue-White DPX E34250ER-Tracker Red™ E34251ER-Tracker Green™ S34200SelectFX™ Alexa Fluor® 488 Endoplasmic Reticulum Labeling Kit B7449Brefeldin A, BODIPY® 558/568 conjugate *isomer 1*

7 ER-Tracker ™ Family: Live Cell Endoplamic Reticulum Markers E12353 ER-Tracker™ Blue-White DPX E34251 ER-Tracker Green™ E34250 ER-Tracker Red™

8 Golgi A21270anti–golgin-97 Ab (Primary Ab or fixed Cells) N22651NBD C 6 -ceramide complexed to BSA B22650BODIPY® FL C 5 -ceramide complexed to BSA B34400BODIPY® TR C 5 -ceramide complexed to BSA

9 Mitochondria M7512MitoTracker® Red CMXRos M7514 MitoTracker® Green FM M7510 MitoTracker® Orange CMTMRos T3168 JC-1 A6403 anti–cytochrome oxidase subunit I (from panel of Ox Phos Complex Primary Ab´s directed against Mitochondrial Enzymes)

10 Lysosomes L7528LysoTracker® Red DND-99 L7526 LysoTracker® Green DND-26 L7545LysoSensor™ Yellow/Blue DND- 160

11 Peroxisomes S34201SelectFX™ Alexa Fluor® 488 Peroxisome Labeling Kit

12 Cytoskeleton/Tubulin T34075TubulinTracker™ Green A11126anti–α-tubulin mouse monoclonal IgG

13 TubulinTracker ™ Green Drastically improved tubulin labeling in living cells Live BPAE cells + Oregon Green Taxol Live BPAE cells + TubulinTracker™ Green

14 Cytoskeleton/Actin A12379Alexa Fluor® 488 phalloidin R415rhodamine phalloidin A12381Alexa Fluor® 594 phalloidin

15 Cytosol C2925 CellTracker™ Green CMFDA C34552 CellTracker™ Red CMTPX C3100MPCalcein, AM NewQtracker Family

16 Three color Qtracker Cell Labeling

17 Plasma Membrane V22885Vybrant® DiI cell-labeling solution V22886Vybrant® DiO cell-labeling solution V22887Vybrant® DiD cell-labeling solution W11262Alexa Fluor® 594 wheat germ agglutinin F35355FM® 1-43FX *fixable analog of FM® 1-43 membrane stain*

18 Lipid Rafts B13950BODIPY® FL C 5 -ganglioside GM1 V34403Vybrant® Alexa Fluor® 488 Lipid Raft Labeling Kit V34404Vybrant® Alexa Fluor® 555 Lipid Raft Labeling Kit V34405Vybrant® Alexa Fluor® 594 Lipid Raft Labeling Kit

19 MitoSOX TM Red Oxidant Selectivity MitoSOX TM Red - Highly Selective Superoxide Detection In Mitochondria of Live Cells

20 MitoSOX Red oxidation prevention by superoxide scavengers in live BPAE cells Buffer for 10 min 5 mM MitoSOX TM Red 10 mM FeTCPP for 10 min 5 mM MitoSOX TM Red Xanthine oxidase pre-oxidation of 5 mM MSR No accumulation in mitochondria MitoSOX™ Red mitochondrial superoxide indicator

21 Cell tracers: membrane probes Classic Example: DiI (pronounced “dye eye”) Diffuse along membranes Useful in living or fixed tissues Long retention time (weeks) Generally not well retained upon fixation Workhorses for neural tracing

22 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

23 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

24 Myelin Staining Protocol Comparisons ™ FluoroMyelin™ is More Efficient

25 FluoroMyelin Green™ and FluoroMyelin™ Red Arbor vita of mouse cerebellum sagittal cryosection labeled with FluoroMyelin™ Red, NeuroTrace™ Green Nissl Stain, and DAPI. Mouse rostral mesencephalon cross-section labeled with FluoroMyelin™ Green, NeuroTrace™ Red Nissl Stain, and DAPI. This combination is sold as the new BrainStain™ Imaging Kit. Single-step dyes requiring only 20 minutes label time

26 BrainStain Kit BrainStain™ Imaging Kit The BrainStain™ Imaging Kit: –DAPI –NeuroTrace™ Red Nissl Stain –FluoroMyelin™ Green Mouse diencephalon cross-section Three Labels in One Step One 20-minute label step… …three-color staining for general anatomical visualization, or use the dyes separately.

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28 28 Navigation Viability and vitality reagents –LIVE/DEAD ® kits Apoptosis Reagents –Apoptosis “timeline” –Annexin V conjugates & kits –Mitochondrial reagents –Vybrant ® FAM caspase assay kits –Metabolic and nuclear changes

29 29 Viability: Cell membrane forms intact barrier Vitality: Cell mediates active processes Viability & Vitality Cytosol Nucleus Cytosol Nucleus Viable (Live)Vital (Live) Nonviable (Dead)Nonvital (Dead) + Carboxy- fluorescein + Propidium iodide How it works

30 30 SYTO ® 9 dye SYTO ® 13 dye SYTO ® 16 dye SYTO ® 25 dye SYTO ® 59 dye SYTOX ® Blue SYTOX ® Green Propidium iodide (PI) 7-AAD TO-PRO ® -3 dye Ethidium homodimers Viability & Vitality: Individual Reagent Examples Esterase CFDAs Calcein AM Oxidation Dihydrorhodamine 123 C 12 -resazurin (Vybrant ® Cell Metabolic Assay) Permeant DNA Dyes (Live & Dead) Impermeant DNA Dyes (Dead) Cell Function Markers ViabilityVitality Mix & match based on spectral overlap

31 31 Vitality by Enzyme / Metabolic Function Laser Cytosol Live Cell Dead Cell Calcein AM Ethidium Homodimer-1 Nucleus AM cleavage “It isn’t easy being green”

32 32 Calcein AM and Ethidium Homodimer-1 Vitality: LIVE/DEAD ® Viability / Cytotoxicity Kit (L3224) Vitality = metabolic activity Rapid assay Detects live and dead cells simultaneously The most popular viability assay kit

33 33 New: LIVE/DEAD ® Cell Vitality Assay Kit (L34951) C 12 resazurin fluorescence SYTOX Green dye fluorescence C 12 resazurin + SYTOX ® Green dye Metabolic activity and viability 488 & 633 nm excitation C 12 resazurin alamarBlue Time (minutes) % fluorescence remaining in cells Jurkat cells induced with 10 μM camptothecin for 4 hours, then stained with the LIVE/DEAD ® Cell Vitality Assay Kit Increased permeability / brighter signals than alamarBlue

34 34 Viability: Kit Examples LIVE/DEAD ® Reduced Biohazard Cell Viability Kit #2 (L23101) Viability = membrane integrity LIVE/DEAD ® Reduced Biohazard Cell Viability Kit #2 Principal LIVE/DEAD ® Sperm Viability Kit (L7011) SYBR ® 14 dye and propidium iodide Healthy cells Aged culture Heat-killed cells

35 35 New: Cell Vitality L34955 LIVE/DEAD® Fixable Violet Dead Cell Stain Kit S34857 SYTOX® Blue Dead Cell Stain Cytoplasmic Labeling Fixable Assay Ideal for work with pathogens Also available in Red, Green, and Blue Nucleic Acid Labeling Excites with 405nm laser Quantitative, single-step dead cell indicator Minimal Spectral Overlap

36 36 New Package Sizes: CellTrace™ Kits C34554 CellTrace™ CFSE Cell Proliferation Kit C34555 CellTrace™ Oregon Green ® SE Initial Intensity 7 th doubling New packagings in easy to use aliquots Track cell divisions over multiple generations Existing product repackaged in “experiment-size” aliquots for ease of use and maximum shelf-life 488 nm excitation Increased divisions = decreased fluorescence

37 37 Convenient CyQUANT Cell Proliferation Assay No washes, extractions or long incubations Samples can be frozen Linear from 50 to 50,000 cells; specific for total nucleic acids An excellent alternative to 3 H-thymidine incorporation assays Quick and Easy Sensitive and Accurate No Radioactivity X New CyQuant NF – No Freeze

38 38 New: Cell Cycle Analysis G 0 /G 1 G 2 /M S Identifies major phases of cell cycle -G 0 /G 1, S, G 2 /M Excellent tool for Violet laser Also Available: V35004 Vybrant ® DyeCycle TM Green V35005 Vybrant ® DyeCycle TM Orange V35003 Vybrant ® DyeCycle TM Violet Stain DNA Content Analysis in LIVING Cells

39 39 Viability & Vitality: LIVE/DEAD ® Kits Sku #Kit NameLive StainDead StainExcitationFix? L3224 LIVE/DEAD ® Viability/ Cytotoxicity Kit Calcein AM Ethidium Homodimer nmNo L7010 LIVE/DEAD ® Cell- Mediated/Cytotoxicity Kit DiOC 18 (3)Propidium Iodide488nmNo L7013 LIVE/DEAD ® Reduced Biohazard Cell Viability Kit #1 SYTO ® 10 dyeReactive Red488nmYes L23101 LIVE/DEAD ® Reduced Biohazard Cell Viability Kit #2 Blue Dye dim Blue Dye bright UVYes L23102 LIVE/DEAD ® Reduced Biohazard Cell Viability Kit #3 Red Dye dim Red Dye bright 488nmYes L23105 LIVE/DEAD ® Reduced Biohazard Cell Viability Kit #4 Green Dye dim Green Dye bright 488nmYes L34955 New LIVE/DEAD® Fixable Violet Dead Cell Stain Kit Violet Dye dim Violet Dye bright 405 nmYes L7011LIVE/DEAD ® Sperm Viability KitSYBR ® 14 dyePropidium Iodide488nmNo LIVE/DEAD ® lineup: pick your colors

40 40 Apoptosis – Programmed Cell Death One result … many avenues

41 41 Timeframes within Camptothecin / Jurkat Model* FunctionReagentRelative Time PS TranslocationAnnexin Membrane Integrity PI, SYTOX ® Green dye Mitochondria Activity Changes Membrane Potential Transition Pore MitoTracker ® Red dye DiOC 2 (3), DiIC 1 (5), JC-1 MTP assay Caspase ActivityCaspase substrates Increase Decrease Partial Change Metabolic ActivityC 12 resazurin * Jurkat cells induced with 10 µM camptothecin Putting it into a framework DNA CondensationHoechst DNA FragmentationTUNEL ASSUMED

42 42 Loss of Membrane Asymmetry: Annexin V Probes to detect plasma membrane changes Annexin V Conjugates –Alexa Fluor ® 350 (346/442) –Alexa Fluor ® 488 (495/519) –Fluorescein (496/519) –Oregon Green ® 488 (496/524) –Alexa Fluor ® 568 (578/603) –Alexa Fluor ® 594 (590/617) –Alexa Fluor ® 647 (650/668) –Allophycocyanin (650/660) –Biotin Vybrant Apoptosis Kits (annexin, viability, vitality)

43 43 Loss of Membrane Asymmetry: Annexin V Untreated cells Jurkat cells stained with Vybrant ® Apoptosis Assay Kit #2 (V13241) Induced with camptothecin Apoptotic Dead Viable Apoptotic Dead Viable Detection of phosphatidyl serine translocation

44 44 Selection Guide for Vybrant ® Apoptosis Assay Kits Kit #Cat #Viable Cell StainApoptotic Cell StainDead Cell StainExcitation Kit #1V13240None Alexa Fluor ® 488 dye - Annexin V SYTOX ® Green488nm Kit #2V13241None Alexa Fluor ® 488 dye - Annexin V PI (Red)488nm Kit #3V13242NoneFITC-Annexin VPI (Red)488nm Kit #4V13243NoneYO-PRO ® -1 dye (Green) YO-PRO ® -1 dye PI (Red) 488nm Kit #5V13244Hoechst dimHoechst (Blue)-BrightPI (Red)UV & 488nm Kit #7V23201Hoechst dim YO-PRO ® -1 dye Hoechst 3342 YO-PRO ® -1 dye PI (Red) UV & 488nm Kit #8V35112None R-Phycoerythrin-Annexin V (Orange) SYTOX ® Green dye488nm Kit #9V35113None Allophycocyanin-Annexin V (Far Red) SYTOX ® Green dye 488nm & 633nm Kit #10V35114C 12 -resauzurin (Red) Allophycocyanin-Annexin V (Far Red) SYTOX ® Green dye 488nm & 633nm Kit #11V35116MitoTracker ® Red dye Alexa Fluor ® 488 dye - Annexin V None488nm

45 45 MitoProbe™ Reagents: Detecting Mitochondrial Changes MitoProbe™ JC-1 Assay Kit (M34152) MitoProbe™ DiOC 2 (3) Assay Kit (M34150) MitoProbe™ DiIC 1 (5) Assay Kit (M34151) MitoProbe™ Transition Pore Assay Kit (M34153) Vybrant ® Apoptosis Assay Kit #11 (V35116) Mitochondria: canary in the coal mine

46 46 JC-1 green fluorescence JC-1 red fluorescence + 50 μM CCCPJurkat cells + 2 μM JC-1 Mitochondrial membrane potential New packaging of classic dye with robust protocol 488 nm excitation – uses all emission VIP Product - Widely Used MitoProbe™ JC-1 Assay Kit (M34152) Decrease of red JC-1 signal in apoptotic cells Classic reagent: optimized for flow cytometry

47 47 MitoProbe™ DiOC 2 (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 Vitality Decrease of red and green DiOC 2 (3) signals in apoptotic cells +CCCP Easy reagent – clear result DiOC 2 (3) on HeLa cells

48 48 MitoProbe™ DiIC 1 (5) Assay Kit (M34151) Events DiIC 1 (5) fluorescence +50 μM CCCP Untreated Jurkat cells stained with 50 nM DiIC 1 (5): 633 nm Excitation Mitochondrial membrane potential 633 nm excitation – allows use of 488 nm emissions for other reagents Detection of mitochondrial changes with a 633nm laser Decrease of far red DiIC 1 (5) fluorescence DiIC 1 (5) on MRC5 cells

49 49 MitoProbe ® Transition Pore Assay Kit (M34153) BPAE cells + calcein AM / CoCl 2 Calcein + Cobalt Calcein + Cobalt + Ionomycin Calcein fluorescence F G H Calcein Alone Calcein fluorescence Whole cell fluorescenceMitochondrial fluorescenceDiminished fluorescence New assay - 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 ABC DE

50 50 MitoProbe ® Transition Pore Assay Kit (M34153) BPAE cells labeled with calcein AM, MitoTracker ® Red dye, and Hoechst AB DC Seeing the assay with your own eyes

51 51 Vybrant ® FAM Caspase Assay Kits Probes to detect caspase activation Vybrant ® FAM Caspase-3 and –7 Assay Kit (V35118) Vybrant ® FAM Caspase-8 Assay Kit (V35119) Vybrant ® FAM Polycaspases Assay Kit (V35117)

52 52 Vybrant ® FAM Caspase Assay Kits: FLICA Function Detection of active caspases in apoptotic cells

53 53 Vybrant ® FAM Caspase Assay Kits Vybrant ® FAM Polycaspases Assay Kit (V35117) Vybrant ® FAM Caspase-8 Assay Kit (V35119) Vybrant ® FAM Caspase-3 and –7 Assay Kit (V35118) FAM Fluorescence Propidium Iodine Fluorescence Dead Cells PI Positive Apoptotic Cells FAM Positive Live Cells FAM Negative Increase of green FAM-FLICA Fluorescence in apoptotic cells Reliable detection of active caspases

54 54 Example of multiparameter analysis APC annexin V fluorescence 30 minutes1 hour 2 hours 3 hours FAM Polycaspase fluorescence Note the shift in APC fluorescence (Green arrow) prior to shift in FAM fluorescence (Blue arrows) Allows study of timing of apoptoic events (I.e. membrane asymmetry prior to caspase activation) Live cells, annexin V-positive, PI-positive Multiparameter: caspase activity & membrane assymetry Easily combine studies of caspase activation with membrane asymmetry FAM-polycaspase Green Channel Propidium Iodine Red Channel APC-Annexin V Far Red Channel

55 55 Detecting Metabolic and Nuclear Changes Vybrant ® Apoptosis Assay Kit #10 (V35114) Vybrant ® Apoptosis Assay Kit #5 (V13244) APO-BrdU ™ TUNEL Assay Kit (A23210) Metabolic & nuclear changes: the end game

56 56 Vybrant ® Apoptosis Assay Kit #10 (V35114) 4 hours camptothecin Necrotic (peroxide- treated) APC annexin VSYTOX ® Green Dye Resorufin fluorescence C 12 -resazurin resorufin Reductase Allophycocyanin annexin V + C 12 resazurin + SYTOX ® Green dye Membrane asymmetry, metabolic activity and membrane integrity 488 & 633 nm excitation AB CD Jurkat cells treated as labeled: 488 nm & 633 nm excitation Live cells, annexin V-positive, SYTOX ® Green-positive Reductase activity / metabolic health

57 57 Chromatin Condensation / DNA Fragmentation Vybrant ® Apoptosis Assay Kit #5 (V13244) Hoechst / propidium iodide APO-BrdU TM TUNEL Assay Kit (A23210) Alexa Fluor ® 488 anti-BrdU / propidium iodide Nuclear changes: the finale

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64 64 Literature and brochures Easy downloads from the web, or available on request.


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