Cd8 v500 rpa t8

Manufactured by BD

Sourced in United Kingdom, United States

The CD8-V500 (RPA-T8) is a fluorochrome-conjugated monoclonal antibody designed for flow cytometric analysis of human CD8 antigen expression on T cells. The CD8 antigen is a cell surface glycoprotein that serves as a co-receptor for the T-cell receptor and is expressed on a subset of T lymphocytes.

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RPA-T8 (53 citations) CD8-V500 (31 citations)

6 protocols using cd8 v500 rpa t8

PBMC Immunophenotyping for T Cell Analysis

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PBMCs were used for staining with fluorochrome-conjugated monoclonal antibodies against cell surface markers for T cell activation, exhaustion, and memory differentiation as described previously.²¹ Briefly, freshly isolated PBMCs were washed in phosphate buffered saline, adjusted to two million cells per stain, and incubated with titrated concentrations of the following antibodies in a staining volume of 50 μL in PBS: 1:100 CD3/BV605 (SK7; BD Biosciences), 1:100 CD4/V450 (RPA-T4; BD Biosciences), 1:100 CD8/V500 (RPA-T8; BD Biosciences), 1:50 PD-1/APC (MIH4; eBioscience), 1:25 CCR7/PE (150503; R&D Systems), 1:50 CD45RA/Alexa Fluor 700 (HI100; BioLegend), 1:25 HLA-DR/FITC (L243; BD Biosciences), and 1:25 CD38/PE-Cy7 (HIT2; BD Biosciences). After 20 min incubation at room temperature cells were washed twice in PBS and resuspended in PBS with 2% paraformaldehyde. Near-infrared LIVE/DEAD marker (Invitrogen) was used to exclude nonviable cells. Fluorescence minus one staining was used to determine the cutoff for PD1 high cells. Data were acquired on a LSR-II driven by FACSDiva software (BD) and analyzed using FlowJo software v8.8.6 (TreeStar, Ashland, OR). All FACS analyses were performed using freshly isolated PBMCs at the HIV Pathogenesis Programme, UKZN, Durban, South Africa.

Muenchhoff M., Healy M., Singh R., Roider J., Groll A., Kindra C., Sibaya T., Moonsamy A., McGregor C., Phan M.Q., Palma A., Kloverpris H., Leslie A., Bobat R., LaRussa P., Ndung'u T., Goulder P., Sobieszczyk M.E, & Archary M. (2018). Malnutrition in HIV-Infected Children Is an Indicator of Severe Disease with an Impaired Response to Antiretroviral Therapy. AIDS Research and Human Retroviruses, 34(1), 46-55.

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Phenotypic Analysis of Activated T Cells

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Single cell suspension of splenocytes, lymph node cells, or thymocytes were made, and these were stained with antibodies CD3-APC (OKT-3, BD Biosciences), CD4-V450 (RM4-5, eBioscience), CD8-V500 (RPA-T8, BD Biosciences company), Vβ8-PE (F23.1, BD Biosciences) KI-67-PerCp-Cy5.5 (BD56, BD biosciences), CD25-PerCp-Cy5.5 (PC61.5, Ebioscience), IFN-γ-FITC (XMG1.2, BD biosciences), CD44-APC (IM7, ebioscience), CD62L-FITC (MEL-14, BD biosciences) or FoxP3-eFluor450 (FJK-16s, ebioscience) and incubated for 30 min at 4°C. Cells were washed three times with PBS containing 2% FCS. Cells were acquired on the FACS Canto II (BD) and analyzed with FlowJo 7 (Tree Star). For cell activation experiments, splenocytes from transgenic mice or littermates were cultured (1 × 10⁵ cells/well) for 24 h in the presence of 20 μg/ml mB29b, in which the last 4 h was in the presence of 1 μg/ml Brefeldin A.

Jansen M.A., van Herwijnen M.J., van Kooten P.J., Hoek A., van der Zee R., van Eden W, & Broere F. (2016). Generation of the First TCR Transgenic Mouse with CD4+ T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide. Frontiers in Immunology, 7, 90.

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PBMCs Phenotyping and Intracellular Staining

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Isolated PBMCs where phenotyped by staining with the following cell surface mAbs: CD3-APC efluor780 (SK7, eBioscience, San Diego, CA), CD4-Qdot655 (S3.5, Invitrogen, Carlsbad, CA), CD8-V500 (RPA-T8, BD Biosciences, Franklin Lake, NJ), and CD14-Pecy7 (M-5E2, BD Biosciences). LIVE/DEAD- Fixable Aqua Stain (Invitrogen, Carlsbad, CA) was used to exclude dead cells from analysis. Intracellular staining for antibodies recognizing CysB-FITC (Assaypro, St. Charles, MO) and CatB-PE (Cell Signaling, Danvers, MA) was performed after permeabilizing and fixing cells with the BD Cytofix/Cytoperm kit according to the manufacturer’s protocols. Supplemental Digital Content Figures 9 and 10 illustrate the gating strategy used. All antibody-stained cells were fixed in 1% formaldehyde (Sigma, St Louis, MO) prior to sample acquisition on a LSR II flow cytometer (BD Biosciences). We ran at least 100,000 gated lymphocytes/monocytes for each stained specimen. Gates for flow cytometric acquisition and analyses were based on isotype controls and single stain compensation controls. Antibody expression was measured via mean fluorescent intensity (MFI). Data were analyzed using FlowJo Version 9.9 software for Mac (TreeStar, San Carlos, CA). Cell viability was >70% across all participant groups.

Opsteen S., Moylan D., Taiwo B.O., Robertson K.R., Overton E.T., Cutter G.R., Sabbaj S., Heath S.L, & Shacka J.J. (2022). Intracellular cystatin B levels are altered in HIV-infected participants with respect to neurocognitive status and anti-retroviral therapy. Journal of acquired immune deficiency syndromes (1999), 91(5), 485-489.

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Isolation and Expansion of Human Regulatory T Cells

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Human ex vivo CD127^loCD25⁺CD4⁺ Tregs were flow sorted using a BD FACSAria cell sorter (BD Biosciences) after staining with: CD39-BB515 (clone Tu66, BD), CD25-PE (clone MA251, BD) or CD25-PE (BD), CD4 APCFire/750 (Clone A161A1, Biolegend) or CD4-ECD (Clone SFCI12T4D11, Beckman Coulter), CD73-BV421 (clone AD2, Biolegend), CD8 V500 (RPA-T8, BD), CD3-BV605 (SK7, Biolegend), CD127-AF647 (clone HIL-7R-M21, BD) or CD127-PEcy7 (eBioRDR5, ebioscience/Thermofisher) and CD45RA-BV786 (clone Hi100, BD), then expanded with 500–1000 U/mL human rIL2 (Miltenyi Biotec/Chiron) ±100 nM rapamycin (Miltenyi Biotec) and human Treg expansion kit anti-CD3/CD28 beads (Miltenyi Biotec/Invitrogen), using x-vivo 15 cell medium (Lonza) or RMPI-1640 medium supplemented with L-glutamine, penicillin-streptomycin (both PAA Laboratories, UK), sodium pyruvate (Gibco, UK) and 2–10% human AB pooled serum and media exchanged with fresh IL-2 every 3–5 days. For additional experiments, either memory Tregs (mTregs) were sorted as CD4⁺CD25⁺CD127^loCD45RA⁻, naïve Tregs were sorted as CD4⁺CD25⁺CD127^loCD45RA⁺ or CD39⁻CD73⁻ double negative Tregs were sorted as CD4⁺CD25⁺CD127^loCD39⁻CD73⁻. Beads were removed using MACSibead magnet (Milteny Biotec) prior to phenotyping or use in functional assays. Example sorts shown in Supplementary Figs. 1a and 2b).

Jarvis L.B., Rainbow D.B., Coppard V., Howlett S.K., Georgieva Z., Davies J.L., Mullay H.K., Hester J., Ashmore T., Van Den Bosch A., Grist J.T., Coles A.J., Mousa H.S., Pluchino S., Mahbubani K.T., Griffin J.L., Saeb-Parsy K., Issa F., Peruzzotti-Jametti L., Wicker L.S, & Jones J.L. (2021). Therapeutically expanded human regulatory T-cells are super-suppressive due to HIF1A induced expression of CD73. Communications Biology, 4, 1186.

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Comprehensive T Cell Immunophenotyping

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LIVE/DEAD Fixable Aqua Stain (Invitrogen), excited by violet 405 nm laser and detected by 512 nm emission channel, was used to determine the percentage of dead cells. The T cell phenotype was determined by staining with mAbs: CD3-APC efluor780 (SK7, eBiosciences), CD4-BV786 (SK3, Biolegend), CD8-V500 (RPA-T8, BD Biosciences), and CD28-BV711 (CD28.2, BD Biosciences). Activation status was determined using mAbs: PD-1-PE-eFlour610 (J105, eBioscience), CD38-BUV737 (HB7, BD Biosciences), CD69-FITC (FN50, BD Biosciences), and CD25-BUV395 (2A3, BD Biosciences). Cytolytic profile was determined using mAbs: TNF-α-APC (6401.1111, BD Biosciences), Granzyme B-BV421 (GB11; BD Biosciences), and IFN-γ-PE-cy7 (B27, BD Biosciences). Monoclonal Ab CD14 BUV805 (M5E2, BD Biosciences, Franklin Lake, NJ) was used to exclude monocytes from analysis. Intracellular staining (ICS) was performed using the BD Cytofix/Cytoperm kit according to the manufacturer’s protocols. All antibody-stained cells were fixed in 1% formaldehyde (Sigma) prior to sample acquisition on a Symphony flow cytometer (BD Biosciences). Gates for flow cytometric acquisition and analyses were based on “fluorescence-minus-one” (FMO) controls and single stain compensation controls.

Crawford C.L., Dalecki A.G., Perez M.D., Schaaf K., Wolschendorf F, & Kutsch O. (2020). A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus. Scientific Reports, 10, 8955.

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Multiparametric Phenotyping of Immune Cells

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Stimulated cells were stained with fluorochrome-conjugated monoclonal antibodies to detect surface and intracellular markers. Intracellular staining was performed with Foxp3 Fixation/Permeabilization buffer kit (eBioscience, San Diego, CA, USA), and Fixable Viability eFluor 780 Dye (eBioscience) was used to exclude dead cells. The following antibodies were used: CD4-Alexa Fluor 700 (OKT4, BioLegend), CD8-V500 (RPA-T8, BD Biosciences, San Jose, CA, USA), GrB-Fitc (GB11, BioLegend), IL-2-Pe (MQ1-17H12, BioLegend), IL-4-PeDaz594 (MP4-25D2, BioLegend), CD137-Pe/Cy7 (4B4-1, BioLegend), CD154-Alexa Fluor 647 (24-31, BioLegend), TNF-α-eFl450 (MAb11, eBioscience), IFN-γ-BV650 (4S.B3, BioLegend), CD3-BV785 (OKT3, BioLegend), CD19-BV605 (HIB19, BioLegend), CD45RA-BV605 (HI100, BioLegend), CCR7-PerCP/Cyanine 5.5 (G043H7, BioLegend).
For the absolute quantification of immune cell subsets, total CD19⁺, CD3⁺, CD4⁺, and CD8⁺ T cells numbers were determined in 50 μL of the peripheral blood by flow cytometry as described below by means of CytExpress software (Beckman Coulter, USA). Absolute numbers of HBs-specific T cells were determined based on relative frequencies of HBs-specific T cells and absolute numbers of total CD4⁺ T cells per μL of peripheral blood.

Awad G., Roch T., Stervbo U., Kaliszczyk S., Stittrich A., Hörstrup J., Cinkilic O., Appel H., Natrus L., Gayova L., Seibert F., Bauer F., Westhoff T., Nienen M, & Babel N. (2021). Robust hepatitis B vaccine-reactive T cell responses in failed humoral immunity. Molecular Therapy. Methods & Clinical Development, 21, 288-298.

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