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Cd8 pecy7 rpa t8

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The CD8 PECy7 (RPA-T8) is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 cell surface antigen. It is commonly used in flow cytometry applications to identify and quantify CD8+ T cells in biological samples.

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Lab products found in correlation

7 aminoactinomycin d 7 aad, by BD (2 mentions) Normal mouse igg, by Thermo Fisher Scientific (2 mentions) Anti car apc antibody, by BD (2 mentions) Cd4 fitc rpa t4, by BD (2 mentions) Cd3 apc cy7 ucht1, by Thermo Fisher Scientific (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Facs aria device, by BD (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Rpa t4, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Facsdiva, by BD (1 mentions) Live dead fixable near ir, by Thermo Fisher Scientific (1 mentions) Anti cd4 pe rpat4, by BD (1 mentions) Cd3 percp cy5, by BD (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Il 2 pe, by BD (1 mentions) Cd25 pe, by BD (1 mentions) Ifn γ apc, by BD (1 mentions) Il 17a pe, by BD (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions)

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RPA-T8 (53 citations)

5 protocols using cd8 pecy7 rpa t8

1

Characterizing CAR T-cell Persistence

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These methods apply to the data shown in Figure 2j–k. Cryopreserved patient peripheral blood mononuclear cells (PBMCs) collected one month after CAR T-cell infusion were thawed and then washed once with FACS buffer containing 0.4% BSA and 0.1% weight/volume sodium azide in PBS. Samples were Fc blocked with normal mouse IgG (Invitrogen) and stained with CD3-APC Cy7 (UCHT1, eBioscience), CD4 FITC (RPA-T4, BD), CD8 PECy7 (RPA-T8, BD), 7-amino actinomycin D (7AAD, BD), and anti-CAR-APC antibody (the anti-CAR antibody was generated by Kite Pharma, Inc.). The anti-CAR antibody bound equivalently to the linker in the scFv of both Hu19-CD828Z and FMC63–28Z (Supplementary Figure 3)35 . Samples were acquired using a BD LSR II with Diva software and data was analyzed using FlowJo version 10. The gating strategy is shown in Supplementary Figure 7.
Brudno J.N., Lam N., Vanasse D., Yueh-wei S., Rose J.J., Rossi J., Xue A., Bot A., Scholler N., Mikkilineni L., Roschewski M., Dean R., Cachau R., Youkharibache P., Patel R., Hansen B., Stroncek D.F., Rosenberg S.A., Gress R.E, & Kochenderfer J.N. (2020). Safety and feasibility of anti-CD19 CAR T cells with fully-human binding domains in patients with B-cell lymphoma. Nature medicine, 26(2), 270-280.
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2

BATF Expression in T Cells

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Peripheral blood mononuclear cells (PBMCs) were freshly isolated from 10 mL EDTA anticoagulated venous blood using Ficoll density-gradient centrifugation and resuspended in RPMI-1640 medium (Invitrogen-Gibco, USA), supplemented with 10% heat-inactivated FBS (Invitrogen-Gibco, USA). To investigate the expression of BATF in peripheral blood T cells, PBMCs (1 × 106) were surface stained with CD4-PB (RPA-T4, BD Pharmingen), CD8-PE-Cy7 (RPA-T8, BD Pharmingen) and PD-1-FITC (MIH4, eBioscience). Cells were then fixed and permeabilized with Cytofix/Cytoperm buffer according to the manufacturer's instruction, stained with BATF-APC (687706, R&D Systems). Stained samples were fixed with 2% paraformaldehyde (PFA), and detected on a BD FACS Aria device. Data were analyzed with FlowJo software (Tree Star Inc., USA).
Liu Q., Ou Q., Shen L., Qiu C., Zhang B., Zhang W., Shao L., Gao Y, & Chen Z.W. (2019). BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection. Frontiers in Immunology, 10, 2430.
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3

Multiparametric Flow Cytometry Analysis

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Where indicated, cells were first stained with Live/Dead Fixable near-IR (Invitrogen, Eugene, OR, USA). Human cells were stained with anti-HLA-DR FITC (G46-6), anti-CD11c PE (B-ly6), anti-CD80 PE-Cy7 (L307.4), anti-CD40 APC (5C3), anti-CD86 BV510 (FUN-1), anti-CD123 BV421 (9F5), CD3 FITC (UCHT1), anti-CD4 PE (RPA-T4), or CD8 PE-Cy7 (RPA-T8) (all BD Biosciences). Mouse cells were stained with anti-IL-17 A PE (TC11-18H10), anti-CD8a V450 (53-6.7) anti-CD40 FITC (3/23) anti-CD80 APC (16–10A1), anti-CD11b BV510 (M1/70) (all BD Biosciences); anti- IFN-γ APC (XM61.2), anti-CD11b PE-Cy7 (M1/70), anti-CD4 PE-Cy7 (RM4–5), anti-CD4 PE (RM4–4) was used when anti-CD4+ mAb was used to deplete CD4+ cell, anti-CD45 PerCP (30-F11), anti-CD90.2 PerCP (30-H12) (all BioLegend San Diego, CA); anti-TNF FITC (MP6-XT22), anti-CD90.2 FITC (54-2.1) anti-Granzyme B PE (NGZB), anti-B220 APC (RA3-6BC), anti-CD11c PE-Cy7 (N418), anti-CD90.2 PerCP-eF710 (30-H12), anti-CD11c PE (N418), or anti-MHC-II PE-Cy7 (M5/114.15.2) (all eBioscience, San Diego, CA). Samples were run on a FACSCanto II (BD Biosciences, San Jose, CA, USA), and analyzed using FACS Diva (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland OR). Fluorescence minus one controls were used to define costimulatory molecule expression by DCs stimulated with poly I:C or chlamydial antigen.
Quispe Calla N.E., Vicetti Miguel R.D., Mei A., Fan S., Gilmore J.R, & Cherpes T.L. (2016). Dendritic cell function and pathogen-specific T cell immunity are inhibited in mice administered levonorgestrel prior to intranasal Chlamydia trachomatis infection. Scientific Reports, 6, 37723.
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4

Characterizing CAR T-cell Persistence

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These methods apply to the data shown in Figure 2j–k. Cryopreserved patient peripheral blood mononuclear cells (PBMCs) collected one month after CAR T-cell infusion were thawed and then washed once with FACS buffer containing 0.4% BSA and 0.1% weight/volume sodium azide in PBS. Samples were Fc blocked with normal mouse IgG (Invitrogen) and stained with CD3-APC Cy7 (UCHT1, eBioscience), CD4 FITC (RPA-T4, BD), CD8 PECy7 (RPA-T8, BD), 7-amino actinomycin D (7AAD, BD), and anti-CAR-APC antibody (the anti-CAR antibody was generated by Kite Pharma, Inc.). The anti-CAR antibody bound equivalently to the linker in the scFv of both Hu19-CD828Z and FMC63–28Z (Supplementary Figure 3)35 . Samples were acquired using a BD LSR II with Diva software and data was analyzed using FlowJo version 10. The gating strategy is shown in Supplementary Figure 7.
Brudno J.N., Lam N., Vanasse D., Yueh-wei S., Rose J.J., Rossi J., Xue A., Bot A., Scholler N., Mikkilineni L., Roschewski M., Dean R., Cachau R., Youkharibache P., Patel R., Hansen B., Stroncek D.F., Rosenberg S.A., Gress R.E, & Kochenderfer J.N. (2020). Safety and feasibility of anti-CD19 CAR T cells with fully-human binding domains in patients with B-cell lymphoma. Nature medicine, 26(2), 270-280.
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5

Flow Cytometric Analysis of Intracellular Cytokines

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Cells were first washed once with PBS. Live/Dead Aqua (0.5ul per sample, ThermoFisher) staining was then performed at room temperature. After two washes, cells were then incubated with surface antibody cocktails. To assess intracellular cytokines, PMA (10ng/ml, Sigma-Aldrich), Ionomycin (500ng/ml, Sigma-Aldrich) and Golgi stop (BD bioscience) were added to cells 4 hrs before staining. Cells were then stained with Live/Dead Aqua marker, following which cell-surface marker staining was performed. Cells were then fixed and permeabilized using fixation/permeabilization buffer (BD bioscience). cytokine antibodies were added and incubated for 30 min. Samples were then washed twice and analyzed by FACSVerse (BD Bioscience). Antibodies used for flow cytometry staining in this study include: CD3-PerCp Cy5.5 (SK7, BD Bioscience), CD4-APC-Cy7 (RPA-T4, BD Bioscience), CD8-PE-Cy7 (RPA-T8, BD Bioscience), SSTR3-Alexa 647 (FAB7018R, R&D system), CD25-PE (BC95, BD Bioscience), CD69-APC (FN50, BD Bioscience), GM-CSF-BV421 (BVD2-21C11, BD Bioscience), IL-17A-PE (N49-653, BD Bioscience), IL-2-PE (5344.111, BD Bioscience) and IFNγ-APC (B27, BD Bioscience).
Zhang B., Feng H., Lin H, & Li R. (2024). Somatostatin-SSTR3-GSK3 modulates human T-cell responses by inhibiting OXPHOS. Frontiers in Immunology, 15, 1322670.
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