Cd8 apc rpa t8

Manufactured by BD

CD8-APC (RPA-T8) is a fluorescently labeled antibody that binds to the CD8 cell surface antigen. It is commonly used in flow cytometry applications to identify and quantify CD8+ T cells.

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CD8-APC (clone RPA-T8, Anti-CD8-APC (clone RPA-T8) RPA-T8 CD8-APC

5 protocols using cd8 apc rpa t8

Mtb-specific T cell Responses Ex vivo

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The freshly collected BAL cells were stimulated ex vivo with Mtb-specific antigens, ESAT-6/CFP-10 and Mtb Cell Wall Fraction (BEI Resources, 10 μg/mL) for a total of 16 h. Brefeldin A (0.5 μg/mL, SIGMA) was added 2 h after the onset of stimulation. After stimulation, the cells were stained with LIVE/DEAD fixable Near-IR stain (ThermoFisher) and stained subsequently with the surface antibodies: CD4-PerCP-Cy5.5 (L200, BD Biosciences), CD8-APC (RPA, T8, BD Biosciences), CD3-AlexaFlour 700 (SP34 2, BD Biosciences), CD95-BV421 (DX2, BD Biosciences), CD28-PECy7 (CD28.2, BD Biosciences) and CD45-BUV395 (D058 1283, BD Biosciences). Cells were then fixed, permeabilized and stained with intracellular antibodies: IFNγ-APC-Cy7 (B27, Biolegend), IL-17-BV605 (BL168, Biolegend) and TNF-α-BV650 (MAb11, Biolegend). Cells were washed, suspended in BD stabilizing fixative buffer and acquired on BD Symphony flow cytometer. Analysis was performed using FlowJo (v10.6.1) using previously published gating strategy (18 (link)–20 (link)) (Figures S1–S3).

Sharan R., Singh D.K., Rengarajan J, & Kaushal D. (2021). Characterizing Early T Cell Responses in Nonhuman Primate Model of Tuberculosis. Frontiers in Immunology, 12, 706723.

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Multiparameter Flow Cytometry Panels

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The following Abs were used for culture or surface and intracellular cytokine staining for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-PECY7 (SP34-2, BD), CD3-PB (SP34-2, BD), CD4-APC (L200, BD, used for measuring CD4 expression after CD4 depleting Ab treatment), CD8-PB (RPA-T8, BD), CD8-APC (RPA-T8, BD), CD8-PE (RPA-T8, BD), IFN-γ-Allophycocyanin (4S.B3, BD), IFN-γ-PE (4S.B3, BD), TNF-α-PE (MAb11, BD), TNF-α-APC (MAb11, BD), TNF-α-PB (MAb11, eBioscience), IL-17-PE (eBio64CAP17, eBioscience), IL-17-Alexa647 (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD, see reference(44 (link))), Streptavidin-Pacific blue (invitrogen). IL-4-APC (8D4-8, eBioscience), Perforin-biotinylated (Pf-344, Mabtech)

Yao S., Huang D., Chen C.Y., Halliday L., Wang R.C, & Chen Z.W. (2014). CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 192(5), 2120-2132.

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T cell and NK cell immunophenotyping

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Whole blood was lysed and labeled with a combination of the following monoclonal antibodies: MHC I-PE-Cy7 (G46-2.6, BD Biosciences, San Jose, CA), CD3-PerCP-Cy5.5 (SP34-2, BD Biosciences), CD4-BV510 (L200, BD Horizon, Franklin Lakes, NJ), CD4-APC (L200, BD Pharmingen, Franklin Lakes, NJ), CD8-APC (RPA-T8, BD Biosciences), CD8-BV421 (RPA-T8, BD Biosciences), CD95-PE (DX2, BD Biosciences), CD28-Pacific Blue (CD28.2, BioLegend, San Diego, CA). Samples were acquired using FACSCanto II or LSRFortessa (BD Bioscience). Samples were analyzed using FCS Express (De Novo Software, Glendale, CA). CD4+ and CD8+ T cells were defined as CD20-CD3+CD4+ or CD20-CD3+CD8+ cells, respectively. NK cells were defined as CD20-CD3-CD56+CD8+ cells.^{13 (link),14 (link)} Naïve T cells were defined as CD28+CD95-. Central and effector memory T cells were defined as CD28+CD95+ and CD28-CD95+, respectively.

Alonso-Guallart P., Duran-Struuck R., Zitsman J.S., Sameroff S., Pereira M., Stern J., Berglund E., Llore N., Pierre G., Lopes E., Kofman S.B., Danton M., Sondermeijer H.P., Woodland D., Kato Y., Ekanayake-Alper D.K., Iuga A.C., Wuu C.S., Wu A., Lipkin W.I., Tokarz R., Sykes M, & Griesemer A. (2020). Impact of CMV reactivation, treatment approaches and immune reconstitution in a nonmyeloablative tolerance induction protocol in Cynomolgus macaques. Transplantation, 104(2), 270-279.

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Comprehensive Immune Cell Profiling

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We performed flow cytometry analysis using the following antibodies: CD3-Percp-Cy5.5 (HIT3a, BD Bioscience), CD8-APC (RPA-T8, BD Bioscience), CD27-PE (M-T271, BD Bioscience), CD28-APC (CD28.2, BD Bioscience), CD45RO-PE (UCHL1, BD Bioscience), CD62L-FITC (DRGE-56, BD Bioscience), αvβ3-FITC (LM6090, EMD Millipore,), αvβ5-FITC (P1F6, EMD Millipore), and NRP-1-PE (AD5-17F6, Miltenyi Biotec GmbH). All samples tested were suspended in FACS buffer and stained with indicated antibodies for 30 min in 4 °C in darks, and then, washed twice, and resuspended in FACS buffer before analysis. For IFN-γ detection, BD™ Cytometric Bead Array (CBA) Human IFN-γ kit (BD Bioscience, USA) was used. Samples were analyzed with BD Accuri C6 (BD Bioscience).

Ding N., Zou Z., Sha H., Su S., Qian H., Meng F., Chen F., Du S., Zhou S., Chen H., Zhang L., Yang J., Wei J, & Liu B. (2019). iRGD synergizes with PD-1 knockout immunotherapy by enhancing lymphocyte infiltration in gastric cancer. Nature Communications, 10, 1336.

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Multiparametric Phenotyping of Immune Cells

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The following flow cytometry antibodies were used: CD11c APC-Vio770 (MJ4-27G12, Miltenyi Biotec), CD14-PerCP (TUK4, Miltenyi Biotec), CD86-phycoerythrin (PE)-Cy7 (2331, BD Biosciences), CD80-PE (L307.4, BD Biosciences), HLA-ABC-VioBlue (REA230, Miltenyi Biotec), HLA-DR/DP/DQ-fluorescein isothiocyanate (FITC) (Tu39, BD Biosciences), CD3-PerCP (SK7, BD Biosciences), CD56-PE (B159, BD Biosciences), CD8-APC (RPA-T8, BD Biosciences) CD107a-FITC (H4A3, BD Biosciences), CD107b-FITC (H4B4, BD Biosciences), CD69-FITC (FN50, BD Biosciences), MICA/B-PE (6D4, BD Biosciences), ULBP2/5/6 (FAB1298p, R&D Systems), IFNγ-BV421 (4S.B3, BD Biosciences), mouse immunoglobulin G1 (IgG1) κ isotype control (PE/FITC/PerCP/PE-Cy7/APC; MOPC-21, BD Biosciences), mouse IgG2a κ isotype control (FITC/PE; G155-178, BD Biosciences), and REA Control-VioBlue (REA293, Miltenyi Biotec).

Jennings V.A., Scott G.B., Rose A.M., Scott K.J., Migneco G., Keller B., Reilly K., Donnelly O., Peach H., Dewar D., Harrington K.J., Pandha H., Samson A., Vile R.G., Melcher A.A, & Errington-Mais F. (2019). Potentiating Oncolytic Virus-Induced Immune-Mediated Tumor Cell Killing Using Histone Deacetylase Inhibition. Molecular Therapy, 27(6), 1139-1152.

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