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Anti cd8 v500

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Anti-CD8-V500 is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 antigen expressed on the surface of T cells. It is designed for use in flow cytometry applications to identify and analyze CD8-positive cell populations.

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Lab products found in correlation

Cytofix cytoperm, by BD (3 mentions) Anti ccr7 pe cy7, by BD (3 mentions) Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (2 mentions) Anti granzyme b pe cf 594, by BD (2 mentions) Anti cd107a pacific blue, by BD (2 mentions) Anti cd4 apc cy7, by BD (2 mentions) Anti cd3 alexafluor700, by BD (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Anti cd3 pacific blue, by BD (2 mentions) Anti tnfα pecy7, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Anti cd14 percp, by BD (2 mentions) Anti cd3 alexa 780, by Thermo Fisher Scientific (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Anti cd4 qdot655, by Thermo Fisher Scientific (2 mentions) 7 aad, by BD (2 mentions) Cell dyn sapphire hematology system, by Abbott (2 mentions) Anti cd15 pecy7, by BD (2 mentions) Anti cd45ro bv421, by BD (2 mentions) Facscanto 2, by BD (2 mentions)

Spelling variants of this product

Anti-CD8 (193 citations) CD8-V500 (31 citations) V500-coupled anti-CD8 (2 citations) V500-conjugated anti-CD8 (53-6,7; (2 citations) V500 mouse anti-human CD8 (2 citations)

18 protocols using anti cd8 v500

1

Comprehensive T-cell Phenotyping Protocol

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Following in vitro re-stimulation, cells were surface-stained with fluorochrome-labeled antibodies anti-CD3-Alexa-Fluor700, anti-CD8-V500 and anti-CD4-APC Cy7 (BD Biosciences, BioLegend). In addition, HLA class I CMV tetramers-PE labeled (A*01 VTE, A*02 NLV, B*07 TPR, B*08 ELR and B*035 IPS) (Beckman Coulter or IBA Solution for Life Sciences), and Live/Dead Fixable Blue Dead-Cell Stain (Invitrogen) was used for gating on viable cells. In some experiments we used surface-stained with fluorochrome-labeled antibodies anti-PD1-FITC and anti CD160-PE Cy7 (BD Biosciences, BioLegend). Cytofix/Cytoperm (BD Biosciences) reagents were used to fix and permeabilize cells for intracellular cytokine staining (ICS) using anti-IFN-γ-PE Cy7 (or BV605), anti-IL-2-FITC, anti-Granzyme B-PE CF 594, anti-CD107a-Pacific Blue, anti-T-bet-PE or BV655 (BD Biosciences) and anti-Eomes-Alexa-Fluor 660 (eBiosciences).
Popescu I., Pipeling M.R., Shah P.D., Orens J.B, & McDyer J.F. (2014). T-bet:Eomes Balance, Effector Function and Proliferation of CMV-specific CD8+ T-cells during Primary Infection Differentiates the Capacity for Durable Immune Control. Journal of immunology (Baltimore, Md. : 1950), 193(11), 5709-5722.
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2

Phenotypic Characterization of Immune Cells

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The following monoclonal antibodies (MAbs) were used for phenotypic analysis: (i) anti-CD27-APC-eFluor780, anti-CD45RA-phycoerythrin (PE), and anti-CD127-eFluor450 (eBioscience, San Diego, CA); (ii) anti-CD3-Pacific Blue, anti-CD8-AmCyan, anti-CD8-V500, anti-CD11a-fluorescein isothiocyanate (FITC), anti-CD95-PE, anti-Ki67-FITC, and anti-CCR7-PE-Cy7 (BD Biosciences, Heidelberg, Germany); (iii) anti-CCR7-FITC (R&D Systems); and (iv) anti-PD-1-PE-Cy7 (BioLegend, San Diego, CA). 7-Aminoactinomycin D (7-AAD; Viaprobe; BD Biosciences) was used for the exclusion of dead cells. All samples were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR).
Nitschke K., Flecken T., Schmidt J., Gostick E., Marget M., Neumann-Haefelin C., Blum H.E., Price D.A, & Thimme R. (2014). Tetramer Enrichment Reveals the Presence of Phenotypically Diverse Hepatitis C Virus-Specific CD8+ T Cells in Chronic Infection. Journal of Virology, 89(1), 25-34.
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3

Multiparametric Flow Cytometry for Immune Profiling

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Following in vitro re-stimulation, cells were surface-stained with fluorochrome-labeled antibodies anti-CD3-Alexa-Fluor700, anti-CD8-V500 and anti-CD4-APC Cy7 (BD Biosciences) and Live/Dead Fixable Blue Dead-Cell Stain (Invitrogen) was used for gating on viable cells. Cytofix/ Cytoperm (BD Biosciences) reagents were used to fix and permeabilize cells for ICS using anti-IFN-γ-BV605, anti-TNF-α-PE-Cy7, anti-IL-2-BV650, anti-Granzyme B-PE CF 594, anti-CD107a-Pacific Blue and anti-T-bet-PE (BD Biosciences).
Popescu I., Pipeling M.R., Mannem H., Shah P.D., Orens J.B., Connors M., Migueles S.A, & McDyer J.F. (2015). Interleukin 12-dependent Cytomegalovirus-Specific CD4+ T cell Proliferation, T-bet induction and Effector Multi-function during Primary Infection are Key Determinants for Early Immune Control. Journal of immunology (Baltimore, Md. : 1950), 196(2), 877-890.
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4

Polyfunctional T-cell Immune Response Analysis

Cited 1 time
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Flow cytometry based ICS assay was done as previously described [35 ,49 (link),50 (link)]. In brief, 106 PBMCs were pulsed with peptide at 10uM in the presence of co-stimulatory antibodies (anti-CD28 and anti-CD49D) and anti-CD107a-FITC (all from BD Biosciences) for 2 hrs at 37°C. Monensin and brefeldin A (both from BD Biosciences) were then added and the cultures incubated for an additional 12 hrs. Next day, the cells were labeled with LIVE/DEAD cell dye (Invitrogen) and surface stained with anti-CD3-Pac Blue, anti-CD8-V500, and anti-CD4-Alexa 780 (both from BD Biosciences). The cells were permeabilized and labeled with anti-IFN-γ-Alexa 700, anti-IL-2-APC, anti-TNFα-PECy7, and anti-Granzyme A-PE (all from BD Biosciences). CD3 events greater than 100,000 were acquired on an LSR II (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar). Polyfunctionality analysis was performed using Boolean gating and SPICE and Pestle software (version 5.1; NIAID).
Erdmann N., Du V.Y., Carlson J., Schaefer M., Jureka A., Sterrett S., Yue L., Dilernia D., Lakhi S., Tang J., Sidney J., Gilmour J., Allen S., Hunter E., Heath S., Bansal A, & Goepfert P.A. (2015). HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses. PLoS Pathogens, 11(8), e1005111.
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5

Multiparametric Flow Cytometry Profiling of Immune Cells

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The following antibodies were used for flow cytometric analysis: Anti-Perforin-Alexa fluor 488, anti-CD28-allophycocyanin (APC), anti-CD19-APC, anti-CD3-APC, anti-CD56-APC, anti-CD3-APC-cyanin7 (Cy7), anti-CD8-APC-Cy7, anti-CD14-APC-Cy7, anti-CD14-fluorescein isothiocyanate (FITC), anti-HLA-DR-FITC, anti-CD16-R-phycoerythrin (PE), anti-GATA3-PE, anti-CD45RA-PE-cyanin 5 (Cy5), anti-CD4-PE-Cy5, anti-CD16-PE-Cy5, anti-CCR7-PE-Cy7, anti-CD4-PE-Cy7, anti-CD4-V450, anti-GranzymeB-V450, anti-CD8-V500, anti-CD3-V500 (all from BD Bioscience, Franklin Lakes, NJ), anti-CD57-FITC, anti-CD4-FITC, anti-CD85j-PE, anti-CX3CR1-PE, anti-T-bet-PE-Cy7, anti-Eomes-Peridinin chlorophyll (PerCP)-efluor710 (six from eBioscience, San Diego, CA), anti-HLA-DR-PE-Cy5, anti-IL-7Rα-V450, anti-CD57-V450 (three from BioLegend, San Diego, CA), anti-CX3CR1-FITC (MBL International Corporation, Woburn, MA). For intracellular staining of T cell lineage-specific transcription factors (T-bet, Gata3, and Eomes), granzyme B and perforin, PBMC were fixed and permeabilized with Fix/Perm buffer set (BioLegend). Stained cells were acquired by a BD LSRFortessa (BD bioscience) and analyzed by using FlowJo software (ver. 9.0 or 10.0; Tree Star, OR).
Kim H.Y., Yoo T.H., Hwang Y., Lee G.H., Kim B., Jang J., Yu H.T., Kim M.C., Cho J.Y., Lee C.J., Kim H.C., Park S, & Lee W.W. (2017). Indoxyl sulfate (IS)-mediated immune dysfunction provokes endothelial damage in patients with end-stage renal disease (ESRD). Scientific Reports, 7, 3057.
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6

CD8 T Cell-Dendritic Cell Interaction

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Epitope-specific CD8 T cells were added directly to autologous iDC at 3:1 effector to target (E/T) ratio in the presence or absence of peptide of interest (10 μM) and co-cultured for 48 hours. Immature dendritic cells cultured in the presence of a maturation cocktail containing TNFα (50ng/ml), IFNα (3000U/ml), IFNγ (1000U/ml), IL-1B (25ng/ml), and pI:C (20ug/ml) were used as a positive control. After two days, cells were stained with dead cell dye (Invitrogen), anti-CD3-Pacific Blue, anti-CD8-V500, anti-CD14-alexa700, anti-CD83-PE, and anti-CD86-FITC (all from BD Pharmingen) at 4°C for 30 min. Cells were then washed and events were acquired on an LSR II flow cytometer.
Qin K., Boppana S., Du V.Y., Carlson J.M., Yue L., Dilernia D.A., Hunter E., Mailliard R.B., Mallal S.A., Bansal A, & Goepfert P.A. (2019). CD8 T cells targeting adapted epitopes in chronic HIV infection promote dendritic cell maturation and CD4 T cell trans-infection. PLoS Pathogens, 15(8), e1007970.
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7

Leukocyte BST2 Expression Analysis

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Surface BST2 expression was detected on different subsets of leukocytes, defined by anti-CD3-Alexa700, anti-CD4-Pacific Blue, anti-CD8-V500, anti-CD14-PerCP (all from BD Biosciences, Heidelberg, Germany) and anti-CD45-FITC (Miltenyi, Bergisch-Gladbach, Germany), by using an anti-BST2-APC conjugated antibody (RS38E, Biolegend). Labeled cells were fixed with 3 % formalin and analyzed on a BD LSRII flow cytometer (BD Biosciences, Heidelberg, Germany). The data files were evaluated using FlowJo Version 8.7 (Tree Star, Ashland, USA). Median fluorescence intensity (MFI) for granulocytes, monocytes and lymphocytes were determined.
Mussil B., Javed A., Töpfer K., Sauermann U, & Sopper S. (2015). Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys. Retrovirology, 12, 92.
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8

Comprehensive Flow Cytometry Analysis of Immune Cell Subsets

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Flow cytometry analysis was done on either heparinized whole blood or cryopreserved PBMCs. For whole blood assays, cells were stained with anti-CD4 APC-H7, anti-CD8 V500 and anti-CD3 PerCP for 20 min at room temperature followed by erythrocyte lysis using BD FACS lysing solution (BD Biosciences). Cells were then fixed and permeabilized with BD Cytofix/Cytoperm for 30 min on ice and then anti-Ki67 FITC and anti-Bcl6 PE added for 30 min on ice in BD permeabilization buffer. To measure blood follicular T helper like cells, cryopreserved cells were resuscitated and stained with anti-CD45RA FITC, anti-PD-1 PE, anti-ICOS PerCP, anti-CXCR5 Alexa 647, anti-CD4 APC-H7, anti-CXCR3 PE-Cy7, anti-CD3 Alexa 700 and live/dead aqua (Invitrogen). All antibodies except anti-PD-1 PE (Biolegend) and anti-CD3 Alexa 700 (Invitrogen) were purchased from BD. To determine the frequency of innate cell populations and their activation status, flow cytometry analysis was done on cryopreserved PBMCs. Following live/dead staining (Alexa Fluor 430, Life Technologies), surface staining of cells was done with an appropriate antibody cocktail. All samples were run on an LSR II (BD Biosciences) and analyzed via FlowJo software. Each cell population was quantified as percentage of total PBMC.
Li S., Sullivan N.L., Rouphael N., Yu T., Banton S., Maddur M.S., McCausland M., Chiu C., Canniff J., Dubey S., Liu K., Tran V., Hagan T., Duraisingham S., Wieland A., Mehta A., Whitaker J.A., Subramaniam S., Jones D.P., Sette A., Vora K., Weinberg A., Mulligan M.J., Nakaya H.I., Levin M., Ahmed R, & Pulendran B. (2017). Metabolic Phenotypes Of Response to Vaccination in Humans. Cell, 169(5), 862-877.e17.
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9

PBMC IFNγ ELISPOT Assay with Peptide Stimulation

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PBMCs responding to both NAE and AE in an IFNγ ELISPOT assay were pulsed with the peptides at 10μM in the presence of anti-CD28 and anti-CD49d. Monensin and brefeldin A were added 1 hour after peptide stimulation. The cells were incubated for an additional 11h. Following incubation, cells were surface stained for 30min at 4°C with dead cell dye (Invitrogen), anti-CD3-Alexa 780 (eBioscience), anti-CD4-Qdot655 (Invitrogen), and anti-CD8-V500 (BD Pharmingen) in the following panels: (1) anti-TIGIT-Percp/CY5.5 (Biolegend), anti-CD160-Alexa488 (eBioscience), anti-PD1-Alexa700 (Biolegend), anti-TIM3-BV421 (Biolegend) and anti-LAG3-PECy7 (Biolegend) and (2) anti-CD28-FITC (BD Pharmingen), anti-CD27-PECy7, anti-CD38-v450 (eBioscience), anti-CD57- Percp/CY5.5 (Biolegend), and anti-CD69-Alexa700 (Biolegend). The cells were then permeabilized and stained with anti-IFNγ-PE at 4°C for 30 min. At least 106 total events were acquired on an LSR II flow cytometer (BD Immunocytometry Systems), and analyzed using FlowJo (version 9.6.4; TreeStar Inc.). The criteria of positivity is the same as defined above for tetramer staining [73 (link)].
Qin K., Boppana S., Du V.Y., Carlson J.M., Yue L., Dilernia D.A., Hunter E., Mailliard R.B., Mallal S.A., Bansal A, & Goepfert P.A. (2019). CD8 T cells targeting adapted epitopes in chronic HIV infection promote dendritic cell maturation and CD4 T cell trans-infection. PLoS Pathogens, 15(8), e1007970.
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10

Phenotyping Immune Cells in NOD Mice

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Thymocytes and peripheral T lymphocyte subsets from NOD, with or without MK626 treatment, were stained using an optimized panel of fluorochrome-conjugated monoclonal antibodies (mAb) (shown in Table 1). Specifically for T cell regulatory subset, T cells from spleen and PLNs were stained with anti-CD4-APCCy5.5 (BD), anti-CD25-PerCP (BD) and anti-FoxP3-eF450 (EBiosciences). Also T cells were stained with anti-CD8-V500 (BD), anti-PD1-PeCy7 (BD) and anti-CD122-biotin (BD). SA-APC (BD) was used as a secondary antibody. Analyses were run on a FACS Canto II flow cytometer (BD).
Alonso N., Julián M.T., Carrascal J., Colobran R., Pujol-Autonell I., Rodriguez-Fernández S., Teniente A., Fernández M.A., Miñarro A., Ruiz de Villa M.C., Vives-Pi M, & Puig-Domingo M. (2015). Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8+T Effector Memory Subset. PLoS ONE, 10(11), e0142186.
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