multicolor-flow-cytometry-support-products-brochure.pdf

full spectrum cell analysisMulticolor flow cytometry Support ProductsTable of ContentsMulticolor Flow Cytometry 1Support Products.11.Proliferation Tracking Dyes 2Cell Proliferation Dye eFluor 670.2CFSE .32.Cell Viability Dyes 4Fixable Viability eFluor Dyes.4Calcein AM,Calcein Blue AM,Calcein Violet 450 AM.5Propidium Iodide&7-AAD.63.Fc-Blocking Reagents 7Improve Your Flow Data.74.Flow Cytometry Buffers&Solutions 81-Step Fix/Lyse Solution(10X).8eBioscience Intracellular Staining Buffers.95.Second Step Reagents 10full spectrum cell analysiseBioscience is committed to developing and manufacturing high-quality,innovative reagents in an ISO certified facility.As a provider of more than 10,000 products,we empower our customers worldwide to obtain exceptional results by using reagents that offer a new standard of excellence in the areas of innovation,quality and value.For Research Use Only.Not for use in diagnostic or therapeutic procedures.eFluor is a registered trademark of eBioscience,Inc.Alexa Fluor is a registered trademark of,and licensed under patents assigned to Molecular Probes,Inc.(Life Technologies).Cy,including Cy5,Cy5.5 and Cy7,is a trademark of Amersham Biosciences Ltd.(GE Healthcare).1Multicolor Flow CytometrySupport ProductseBioscience is an industry-leading provider of Flow Cytometry antibodies against both novel and well-established targets.With the eFluor brand of fluorochromes,including eFluor Nanocrystals,eFluor Organic Dyes and eFluor Functional Dyes,eBioscience offers the broadest and most robust selection of fluorochromes available for multicolor flow cytometry.In addition to these innovative fluorochromes,eBioscience also offers a complete set of flow cytometry support products everything from viability dyes,which help you produce the most accurate data,to staining buffers and secondary antibodies.eBioscience provides a complete solution for your entire flow cytometry workflow.The eFluor brand consists of the following product lines:eFluor NANOCRYSTALS(NC)Direct conjugates for flow cytometry Narrow emission spectra and excellent photostability Expands the utility of violet laser flow cytometry eFluor ORGANIC DYES Direct conjugates for flow cytometry Robust performance for all work flow scenarios eFluor FUNCTIONAL DYES Small molecule organic dyes Viability dyes for intracellular staining protocols Proliferation dyes for tracking cell division Fluorescent dyes to monitor intracellular free calcium2Proliferation Tracking DyeseBioscience provides dyes for analyzing cell proliferation by flow cytometry.With the introduction of Cell Proliferation Dye eFluor 670,detected in the APC channel,you can now monitor cell division in GFP-expressing cells.Cell Proliferation Dye eFluor 670CFSECell Proliferation Dye eFluor 670 Can be used in the presence of FITC or GFP Can be used both in vivo and in vitro Detects up to six rounds of cell division Detected in the APC channelCell Proliferation Dye eFluor 670 is an alternative to CFSE for tracking cell proliferation in situations where CFSE cannot be used due to the presence of FITC or GFP.Cell Proliferation Dye eFluor 670 emits at 670 nm and is excited with the red laser.As with CFSE,Cell Proliferation Dye eFluor 670 accurately indicates as many as six rounds of division by partitioning equally into daughter cells during successive cell division and is useful for both in vitro and in vivo tracking of cell proliferation.Tracking Cell ProliferationLeft:Mouse spleen cells were labeled with 5 M Cell Proliferation Dye eFluor 670,then cultured for three days with(purple histogram)or without(blue histogram)plate-bound anti-mouse CD3 and soluble anti-mouse CD28.Cells were then stained with FITC anti-mouse CD4(cat.no.11-0042)and 7-AAD(cat.no.00-6993).Viable CD4+cells were used for analysis.Right:C57Bl/6 spleen cells were labeled with 5 M Cell Proliferation Dye eFluor 670,then injected into C57Bl/6 x DBA/2 F1 mice.Two days later,spleen cells from the F1 mice were stained with PE-Cy7 anti-mouse CD4(cat.no.25-0042)and Fixable Viability Dye eFluor 450(cat.no.65-0863).Viable CD4+cells were used for analysis.%of MaxCell Proliferation Dye eFluor 670in vitroForward ScatterCell Proliferation Dye eFluor 670in vivo13CFSE Gold standard in cell proliferation analysis Use for tracking cell division both in vitro&in vivo Detected in the FITC channelCFSE is the gold standard for analyzing cell proliferation and has also been used in CTL assays and cell motility studies.CFSE readily crosses intact cell membranes.Once inside the cells,intracellular esterases cleave the acetate groups to yield the fluorescent carboxyfluorescein molecule.The succinimidyl ester group reacts with primary amines,crosslinking the dye to intracellular proteins.Cell division can be measured as successive halving of the fluorescence intensity of CFSE.Tracking Cell Proliferation in vitro T-cell receptor transgenic,OVA-reactive OT-I cells undergo several rounds of cell division in response to OVA,but not PBS,in vivo.Lymph node cells from OT-I mice were labeled with 1 M CFSE(cat.no.65-0850)and adoptively transferred into C57Bl/6 mice.Mice were then immunized with OVA or PBS.Three days later,splenocytes were stained with PerCP-Cy5.5 anti-mouse/rat Thy1.1 and eFluor 450 anti-mouse CD8 to gate on the OT-I cells(left).Cells in the OT-I gate(CD8+Thy1.1+)were analyzed for cell division(right),measured as discrete peaks of decreasing fluorescence of CFSE in OVA-immunized mice(purple histogram),or undivided cells with a single,bright CFSE peak in PBS-immunized mice(blue histogram).Proliferation Tracking Dyes Ordering InformationDescriptionCatalog NumberSizesExcitation LaserDetectorPeak Emission(nm)CFSE65-0850-841 Pack(5 X 500 g)Blue(488 nm)FITC521Cell Proliferation Dye eFluor 670 65-0840-901 Pack(5 X 400 g)Red(633 nm)APC670CD8 eFluor 450Thy1.1 PerCP-Cy5.5%of MaxCFSENew products are launched regularly.Discover more at www.eB.4Cell Viability DyeseBioscience offers a variety of solutions for assessing cell viability.Our multiple viability dyes provide flexibility when designing your multicolor staining panel.Fixable Viability eFluor Dyes can be used to determine viability in both surface and intracellular flow cytometry staining protocols.Fixable Viability Dye eFluor 450,660 and 780Calcein AM,Calcein Blue AM,Calcein Violet 450 AMPropidium Iodide&7-AADFixable Viability eFluor Dyes Assess viability of cells when staining both surface and intracellular targets Assess viability without intercalating DNA Signal maintained in cryopreserved samples Available for the violet and red lasers Provided in convenient 1 l test sizeeBioscience Fixable Viability eFluor Dyes discriminate live cells from dead cells,yet are fully compatible with intracellular staining protocols.Unlike 7-AAD and Propidium Iodide,Fixable Viability eFluor Dyes are retained by dead cells throughout fixation,permeabilization and even cryopreservation protocols.Using Fixable Viability eFluor Dyes with violet and red lasers is easy;simply label cells prior to antibody staining,then follow normal procedures.The result is a brightly fluorescent dead cell population which can be excluded during data analysis,thereby improving data quality.Note:Excluding dead cells from analysis is a recommended procedure for all intracellular staining protocols including detection of nuclear factors,transcription factors,cytosolic signaling molecules as well as secreted growth factors and cytokines.Detection of Viable Cells in Surface Staining BALB/c thymocytes were uncultured(left)or cultured overnight at 37C(right)and then stained with 1 l of Fixable Viability Dye eFluor 780(cat.no.65-0865)per ml of thymocytes that were resuspended at 5x106/ml in PBS.Total cells were used for analysis.Fixable Viability Dye eFluor 780Forward ScatterFixable Viability Dye eFluor 780Forward Scatter25Detection of Viable Cells in Intracellular StainingeFluor Fixable Viability Dye Ordering InformationDescriptionCatalog NumberSizesExcitation LaserPeak Emission(nm)Fixable Viability Dye eFluor 45065-0863-14100 testsViolet(405 nm)45065-0863-18500 tests(5 X 100 tests)Fixable Viability Dye eFluor 66065-0864-14100 testsRed(633 nm)66065-0864-18500 tests(5 X 100 tests)Fixable Viability Dye eFluor 78065-0865-14100 testsRed(633 nm)78065-0865-18500 tests(5 X 100 tests)IntracellularStaining without Fixable Viability DyeIntracellularStaining with Fixable Viability Dye eFluor 450SSC-ACD8 PerCP-eFluor 710#CellsUnlabeledCD4 FITCROR(t)PECD4+CD8+Mouse ThymocytesIntracellular ROR(t)SSC-ACD8 PerCP-eFluor 710#CellsLabeled:Fixable Viabillity Dye eFluor 450CD4 FITCROR(t)PECD4+CD8+Mouse ThymocytesIntracellular ROR(t)False NegativesCalcein AM,Calcein Blue AM,Calcein Violet 450 AMCalcein labeling dyes easily cross the cell membrane and selectively label live cells for analysis by flow cytometry or fluorescence microscopy.Calcein AM is non-toxic and may be used for short-term cell tracing.Calcein Blue AM,a UV excited alternative to Calcein AM,has excitation characteristics similar to DAPI,Hoechst,and AMCA dyes.Calcein Violet 450 AM is a violet laser(405 nm)equivalent to Calcein AM.Co-staining with Annexin V or 7-AAD is recommended to allow the greatest resolution between live and dead/apoptotic cells.6Calcein Blue AM Balb/c thymocytes were stained with 1 M Calcein Blue AM(cat.no.65-0855)for 30 minutes at room temperature(left).Thymocytes were kept on ice overnight(shaded histogram)or cultured overnight at 37C without(purple)or with(blue)1 M dexamethasone.Thymocytes cultured overnight without dexamethasone were also stained with Annexin V-APC(cat.no.88-8007)allowing further discrimination between live and dead cells(right).Total cells were used for analysis.Calcein Violet 450 AM Balb/c thymocytes were stained with 1 M Calcein Violet 450 AM(cat.no.65-0854)for 30 minutes at room temperature(left).Thymocytes were kept on ice overnight(shaded histogram)or cultured overnight at 37C without(purple)or with(blue)1 M dexamethasone.Thymocytes cultured overnight without dexamethasone were also stained with Annexin V-APC(cat.no.88-8007)allowing further discrimination between live and dead cells(right).Total cells were used for analysis.Calcein Labeling Dyes Ordering InformationDescriptionCatalog NumberSizesExcitation LaserPeak Emission(nm)Calcein,AM65-0853-391 mgBlue(488 nm)51565-0853-781 Pack(20 X 50 g)Calcein Blue,AM65-0855-391 mgUV(355 nm)445Calcein Violet 450,AM65-0854-391 mgViolet(405 nm)450%of MaxCalcein Blue AMCalcein Blue AMAnnexin V-APC%of MaxCalcein Violet 450 AMCalcein Violet 450 AMAnnexin V-APCPropidium Iodide&7-AADeBioscience offers Propidium Iodide(PI)and 7-amino-actinomycin D(7-AAD)for assessing cell viability by flow cytometry.Both dyes are available as ready-to-use solutions for the exclusion of nonviable cells in flow cytometric analysis.PI binds to double stranded DNA of dead cells and is excluded from cells with intact plasma membranes.PI can be detected with the PerCP tandem dye channel for viability exclusion.Analyze PI in the PE channel when used as a counterstain for FITC-Annexin V.7-AAD can be used in place of PI.Fluorescence is detected in the far red range of the spectrum(650 nm long-pass filter).PI and 7-AAD can also be used for flow cytometric analysis of the cell cycle.Propidium Iodide&7-AAD Ordering InformationDescriptionCatalog NumberSizesExcitation LaserPeak Emission(nm)Propidium Iodide(PI)00-6990-502 mlBlue(488 nm)6177-AAD65-6993-502 mlBlue(488 nm)6477Fc-Blocking ReagentsImprove Your Flow Data Block non-specific Fc-mediated binding of antibodies Reduce background and improve resolution of flow cytometry dataeBioscience Fc-Blocking reagents are used to inhibit the non-specific Fc-gamma receptor(FcR)-mediated binding of antibodies used for flow cytometric analysis.Blocking non-specific Fc fragment-mediated binding of antibodies to cell surface Fc receptors is necessary during staining for flow cytometry for optimal staining and signal to noise ratio.eBioscience provides reagents for blocking Fc-binding in both mouse and human cells.Four different classes of Fc receptors have been identified and are expressed at varying levels in multiple cell lineages with high expression in myeloid,granulocyte and B cells.The extent to which monoclonal antibodies will bind to FcR varies depending on the isotype of the monoclonal antibody.Furthermore,different monoclonal antibodies of the same isotype will display differential binding to FcRs.Fc-Blocking Reagents Ordering InformationDescriptionCloneIsotypeFormatSizeCatalog NumberHuman FcR-Binding Inhibitor-Purified25 tests14-9161-71-Purified100 tests14-9161-73-Functional Grade25 tests16-9161-71-Functional Grade100 tests16-9161-73anti-mouse CD16/32-BlocksFc-Binding93Rat IgG2a,Purified50 g14-0161-8193Rat IgG2a,Purified100 g14-0161-8293Rat IgG2a,Purified500 g14-0161-8593Rat IgG2a,Purified1 mg14-0161-8693Rat IgG2a,Functional Grade50 g16-0161-8193Rat IgG2a,Functional Grade100 g16-0161-8293Rat IgG2a,Functional Grade500 g16-0161-8593Rat IgG2a,Functional Grade1 mg16-0161-863New products are launched regularly.Discover more at www.eB.8Flow Cytometry Buffers&SolutionsOptimal detection of cellular antigens by flow cytometry requires the use of optimized staining buffers.eBioscience provides appropriate staining and cell preparation buffers regardless of whether your target is secreted,nuclear,or on the cell surface.1-Step Fix/Lyse Solution(10X)eBioscience 1-Step Fix/Lyse Solution simplifies your analysis of human blood.This solution lyses red blood cells(RBCs)after staining peripheral blood with fluorochrome conjugated antibodies,and leaves behind stained and fixed leukocytes,all in one step.1-Step Fix/Lyse Solution Advantages Include:RBC lysis and fixation of samples in only 15 minutes Allows temporary storage of fixed and stained samples Eliminates the need for gradient centrifugation separation1-Step Fix/Lyse Solution(10 x)Normal human peripheral blood cells were stained with FITC anti-human CD45,PerCP-eFluor 710 anti-human CD3,and eFluor 450 anti-human CD19,and then incubated with eBioscience 1-Step Fix/Lyse Solution(cat.no.00-5333)for 15 minutes at room temperature.Cells were centrifuged,washed once in flow staining buffer,and then analyzed.Left:CD45+granulocytes(red),monocytes(blue),and lymphocytes(green)can be seen in the forward vs.side scatter plot of total viable cells.Right:Analysis of cells gated on CD45+events.SSC-AFSC-ACD3 PerCP-eFluor 710CD19 eFluor 4504New products are launched regularly.Discover more at www.eB.9eBioscience Intracellular Staining BuffersWhen performing intracellular staining for flow cytometry the selection of buffers used for fixation and permeabilization has a significant impact on the quality and accuracy of data.eBioscience provides optimized buffer solutions for nuclear factors,transcription factors,and cytosolic and secreted proteins.The table below summarizes which buffer system is appropriate for target antigens in various cellular locations.Intracellular Sta