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Cd4 fitc rpa t4

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CD4 FITC (RPA-T4) is a fluorescent-labeled monoclonal antibody that binds to the CD4 cell surface receptor. It is used for the identification and enumeration of CD4-positive cells in flow cytometry applications.

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Lab products found in correlation

Normal mouse igg, by Thermo Fisher Scientific (2 mentions) Cd3 apc cy7 ucht1, by Thermo Fisher Scientific (2 mentions) Anti car apc antibody, by BD (2 mentions) Cd8 pecy7 rpa t8, by BD (2 mentions) 7 aminoactinomycin d 7 aad, by BD (2 mentions) Permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Foxp3 apc, by Thermo Fisher Scientific (1 mentions) Ki67 pe b56, by BD (1 mentions) Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Facscalibur, by BD (1 mentions) Ifn γ pe cy7 4s b3, by BioLegend (1 mentions) Cd8 percp cy5.5 rpa t8, by BD (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Tnf α pe cy7 mab11, by BioLegend (1 mentions)

Close spelling variants of this product

CD4-FITC (clone RPA-T4, Anti-CD4-FITC (clone RPA-T4) RPA-T4 CD4-FITC

4 protocols using cd4 fitc rpa t4

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

Phenotyping T Regulatory Cells

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PBMC surface antigens were stained with CD4-FITC (RPA-T4), CD25-APC (MA251), Ki67-PE (B56), and CD127-PE (hIL-7R-M21) (BD Bioscience, San Diego, CA). After washing to remove unbound antibody, cells were incubated on ice for 30 minutes in fixation/permeabilization buffer (eBioscience, San Diego, CA). Cells were washed again with 1X permeabilization buffer (eBioscience), pelleted, and stained with FOXP3-APC (PCH101, eBioscience). Samples were acquired on BD FACS Calibur (BD, San Diego, CA) and analyzed with FlowJo (TreeStar, Ashland, OR). TRegs were defined as CD25+FOXP3+ of CD4+ lymphocytes.
Batich K.A., Reap E.A., Archer G.E., Sanchez-Perez L., Nair S.K., Schmittling R.J., Norberg P., Xie W., Herndon JE I.I., Healy P., McLendon R.E., Friedman A.H., Friedman H.S., Bigner D., Vlahovic G., Mitchell D.A, & Sampson J.H. (2017). Long-term Survival in Glioblastoma with Cytomegalovirus pp65-targeted Vaccination. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(8), 1898-1909.
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3

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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4

Activation and Cytokine Profiling of PBMCs

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Peripheral blood mononuclear cells (1 × 106) were stimulated with PMA (500 ng/ml), ionomycin (2 μg/ml), and brefeldin A (10 μg/ml) for 5 h at 37°C in 5% CO2. The surface were stained with CD3-BV421 (UCHT1), CD8-percp-cy5.5 (RPA-T8), and CD4-FITC (RPA-T4) (BD Biosciences), and intracellular staining was performed using anti-IL-17A-PE (N49-653), IL-10-APC (JES3-19F1) (BD Biosciences), IL-4-APC (8D4-8), TGF-β1-PE (TW4-6H10), TNF-α-PE-cy7 (MAb11), and IFN-γ-PE-cy7 (4S.B3) (Biolegend, San Diego, CA, USA) and the foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocols. Unstimulated cells were used as a negative control.
Lu X., Su B., Xia H., Zhang X., Liu Z., Ji Y., Yang Z., Dai L., Mayr L.M., Moog C., Wu H., Huang X, & Zhang T. (2016). Low Double-Negative CD3+CD4−CD8− T Cells Are Associated with Incomplete Restoration of CD4+ T Cells and Higher Immune Activation in HIV-1 Immunological Non-Responders. Frontiers in Immunology, 7, 579.
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