Rpa t8

Manufactured by BD

Sourced in United Kingdom

The RPA-T8 is a laboratory equipment device designed for automated pipetting and liquid handling tasks. It features eight independent channels for simultaneous liquid transfers with adjustable volumes. The device is capable of performing a wide range of liquid handling procedures with high accuracy and precision.

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Lab products found in correlation

Rpa t4, by BD (20 mentions) Ionomycin, by Merck Group (4 mentions) Facscalibur flow cytometer, by BD (4 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions) Facscanto 2, by BD (3 mentions) Cellquest software, by BD (3 mentions) Hit3a, by BioLegend (3 mentions) Lsrfortessa, by BD (3 mentions) Rpa t4, by BioLegend (2 mentions) Live dead near ir stain, by Thermo Fisher Scientific (2 mentions) Lsr 2, by Cytek (2 mentions) Aurora cytometer, by Cytek (2 mentions) Formaldehyde, by Thermo Fisher Scientific (2 mentions) Anti cd3 fitc, by BioLegend (2 mentions) Cd4 bv650, by BD (2 mentions) Anti cd14 apc cy7, by BioLegend (2 mentions) Cd14 apc, by BD (2 mentions) Cd8 apc, by BD (2 mentions) Hiperfect transfection reagent, by Qiagen (2 mentions) Sp34 2, by BD (2 mentions)

Close spelling variants of this product

Anti-CD8 (clone RPA-T8; (8 citations) CD8-V500 (RPA-T8, (6 citations) CD8-APC (RPA-T8, (5 citations) BUV395-anti-CD8 (clone RPA-T8, (5 citations) Anti-human CD8 (clone RPA-T8, (4 citations) CD8 BV711 (clone RPA-T8), (4 citations) Anti-human CD8 (RPA-T8). (4 citations) CD8-BUV395 (RPA-T8, (3 citations) CD8 PacificBlue (RPA-T8, (3 citations) CD8 V450 (RPA-T8; (3 citations)

53 protocols using rpa t8

Identifying MAGE-A4-specific CD8+ T cells

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Staining of cell surface proteins was done according to standard protocols.19 26 (link) Intracellular staining to identify Granzyme B was done using the FoxP3/transcription buffer set (ThermoFisher, 00-5523-00) according to the manufacturer’s protocol. The following fluorochrome-labeled antibodies were used: anti-human CD8 (RPA-T8, Becton Dickinson, 560347), CD4 (RPA-T8, Becton Dickinson, 7137857), Granzyme B (GB11, BioLegend, 515405), HLA-A2 (BB7.2, Becton Dickinson, 561341), CD137 (4B4-1, Becton Dickinson, 550890), CD154 (TRAP1, Becton Dickinson, 555700).
CD8⁺ T cells specific for MAGE-A4 in context of HLA-A2 were identified by multimer staining. Fluorochrome-labeled pentamers were custom-synthesized (ProImmune) and staining was done according to the manufacturer’s instructions, also including staining for CD8 and CD4 when appropriate. Cell sorting was conducted using a FACSAria Fusion flow cytometer (Becton Dickinson) or a SH800S cell sorter (Sony).

Davari K., Holland T., Prassmayer L., Longinotti G., Ganley K.P., Pechilis L.J., Diaconu I., Nambiar P.R., Magee M.S., Schendel D.J., Sommermeyer D, & Ellinger C. (2021). Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy. Journal for Immunotherapy of Cancer, 9(3), e002035.

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Multiparametric Flow Cytometry for T-Cell Profiling

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Aliquots (50 μl) of EDTA-treated whole blood were stained with monoclonal antibodies to CD3 PerCP (SP34-2, BD Biosciences, 552851), CD4 FITC (OKT-4, Biolegend, 317408), and CD8 PE (RPA-T8, BD Biosciences, 555367). CD4⁺ T-cell counts were determined with BD Trucount tubes according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, United States). PBMCs were isolated using conventional Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). Polychromatic flow cytometry was performed to quantitate activated CD4⁺ or CD8⁺ T lymphocytes (Figure 2) and CD4⁺ or CD8⁺ memory T lymphocyte subsets (Figure 3). Activated or memory T lymphocyte subsets (Table 1) from 1 × 10⁶ PBMCs were stained with anti-CD3 BV605 (SP34-2, BD Biosciences, 562994), anti-CD4 BV711 (OKT-4, Biolegend, 317440), anti-CD8 PE (RPA-T8, BD Biosciences, 557086), anti-CCR7 BV421 (G043H7, Biolegend, 352208), anti-CD45RA APC (5H9, BD Biosciences, 561210), anti-CD38 FITC (AT-1, Stemcell, 60131FI), anti-HLA-DR BV510 (G46-6, BD Biosciences, 563083), and anti-PD-1 PerCP-cy5.5 (EH12.2H7, Biolegend, 329914) monoclonal antibodies. Cells were resuspended in 1% paraformaldehyde, subjected to flow cytometry within 24 h on a FACSAriaII (BD Biosciences, San Diego, CA, United States), and analyzed using FlowJo V₁₀ software.

Tong L., Cong Z., Tian L., Zhang J., Lu J., Lu Q., Chen T., Wang Y., Wei Q, & Xue J. (2021). Stage-Dependent Within-Individual Comparison Reveals SIV-Specific Activation/Exhaustion Shift in Rhesus Macaques. Frontiers in Microbiology, 12, 704449.

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Multiparameter Flow Cytometry Analysis

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Cells were stained with antibodies targeting surface or intracellular molecules of interest according to manufacturer’s instructions and analyzed on a Fortessa flow cytometer (BD Bioscience). Antibodies against mouse TCR β:Hamster-PE (1:200 dilution, Biolegend, H57-597), human CD8:PacificBlue (1:50 dilution, BD, RPA-T8), human CD8:BB515 (1:100 dilution, BD, RPA-T8), human CD4:PerCP-eF710 (1:100 dilution, eBioscience, SK3), human CD25:PE-Dazzle 594 (1:100 dilution, Biolegend, M-A251), human CD127:Brilliant Violet 650 (1:100 dilution, Biolegend, A019D5) and human FoxP3:APC (1:100 dilution, eBioscience, 236A1E7) were used. The dyes Life/Dead (1:1000 dilution, Themo Fisher) and CTV (1:1000 dilution, Themo Fisher) were used to stain cells.

Zhang Z., Kong X., Ligtenberg M.A., van Hal-van Veen S.E., Visser N.L., de Bruijn B., Stecker K., van der Helm P.W., Kuilman T., Hoefsmit E.P., Vredevoogd D.W., Apriamashvili G., Baars B., Voest E.E., Klarenbeek S., Altelaar M, & Peeper D.S. (2022). RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells. Cell Reports Medicine, 3(6), 100655.

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Assessment of Anti-PGLALA Binding in PBMCs

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Freshly prepared PBMCs (1 × 10⁵/condition) were incubated with 10 μg of rituximab (Roche), pembrolizumab IgG₄ (InvivoGen, 375939001), pembrolizumab‐PGLALA (for comparison reasons, Roche), or isotype controls IgG₁ LEAF (BioLegend, 400166), IgG₄ LEAF (BioLegend, 403702) or DP47 (Roche) for 30 min on ice, then washed twice with PBS. Anti‐PGLALA binding was assessed by flow cytometry after incubation with a PBS based staining master mix (50 μl/condition) containing CD45‐PerCPCy5.5 (1:100, HI30, BioLegend, 304028), CD3‐APC‐Cy7 (1:100, UCHT1, BD, 300426), CD4‐AF647 (1:100, RPA‐T4, BioLegend, 300520), CD8‐AF488 (1:100, BD, RPA‐T8, 557696), CD19‐BV421 (1:100, HIB19, BioLegend, 302220 and anti‐PGLALA‐PE (Roche, 1:160), for 30 min on ice. Cells were then acquired on a BD FACS‐Symphony cytometer (five lasers: 355, 405, 488, 561, 637 nm). Compensation was performed using VersaComp antibody capture beads (Beckman, B22804). PGLALA‐PE expression was assessed on CD3⁺ T cells and CD19⁺ B cells. Refer to Supporting Information for details on anti‐PGLALA‐PE generation.

Junker F., Gulati P., Wessels U., Seeber S., Stubenrauch K., Codarri‐Deak L., Markert C., Klein C., Camillo Teixeira P, & Kao H. (2021). A human receptor occupancy assay to measure anti‐PD‐1 binding in patients with prior anti‐PD‐1. Cytometry, 99(8), 832-843.

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NK Cell Degranulation Assay with GAD65 Peptide

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Degranulation assay of NK cells following GAD65 AA 114–122 peptide presentation was performed through quantification of cell-surface CD107a expression by FACS analysis [40 (link)]. Briefly, at the end of the co-culture period, culture plate was centrifuged at 2000 rpm for 2 minutes and cell staining was directly performed by adding the mixture of mouse mAbs anti- human CD3 Alexa Fluor 700-A (1:50, BD), CD56 PECy7 (1:50, BD), CD8 PECy5 (1:30, clone RPA-T8, cat# 557746, BD), ILT2 (APC,1:50, eBioscience) and CD107a FITC (1:10, clone H4A3, cat# 555800, BD).

Perri V., Gianchecchi E., Cifaldi L., Pellegrino M., Giorda E., Andreani M., Cappa M, & Fierabracci A. (2017). Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers. PLoS ONE, 12(12), e0189615.

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Isolation and Characterization of Ascites Cells

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Cells from the patient ascites fluid were isolated as described previously [31 (link)]. In brief: after filtration of the ascites fluid through a 40 μM mesh, cells were sedimented by 10 min centrifugation at 350 g. Then, cells were fixed and permeabilised using the Human FoxP3 Buffer Set (BD), stained with CD3-Pacific Blue (UCHT1, BD Biosciences, AB_397038), CD4-PerCP (L200, BD Biosciences, AB_393791), CD8-PE-Cy7 (RPA-T8, BD Biosciences, AB_396856) and CD45RA-APC-H7 (HI100, BD Biosciences, AB_1727497) and run on a FACSCanto II (BD Biosciences). Data visualization and analysis was done in FlowJo (V10) (FlowJo, LLC).

Landskron J., Kraggerud S.M., Wik E., Dørum A., Bjørnslett M., Melum E., Helland Ø., Bjørge L., Lothe R.A., Salvesen H.B, & Taskén K. (2017). C77G in PTPRC (CD45) is no risk allele for ovarian cancer, but associated with less aggressive disease. PLoS ONE, 12(7), e0182030.

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Sorting HIV-specific Memory B Cells

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PBMCs were stained as described previously (18 ). Briefly, frozen PBMCs were thawed and treated with 10,000 U/ml DNase I (Roche) in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) media, followed by Aqua Dead Cell Staining (Life Technologies). A cocktail of antibodies containing CD3 (APC-Cy7; SP34-2, BD Pharmingen), CD8 (Pacific blue; RPA-T8, BD Pharmingen), CD14 (Qdot 605; M5E2, VRC), CD20 (PE-Alexa Fluor 700; 2H7, VRC), CD27 (PE-Cy7; M-T271, BD Pharmingen), IgG (FITC; G18-145, BD Pharmingen), and IgM (PE-Cy5; G20-12, BD Pharmingen) was used to stain the PBMCs. To sort Env and CD4bs-specific B cells, gp140-F conjugated with streptavidin-allophycocyanin conjugate (SA-APC, Life Technologies) and gp140-F-D368R with streptavidin-phycoerythrin conjugate (SA-PE, Life Technologies) were incorporated in the above-stated antibody cocktail. Following staining, the cells were sorted at a single-cell density into 96-well plates with lysis buffer using a four-laser FACS Aria III cell sorter. Env-specific memory B cells were defined as CD3⁻CD8⁻Aqua Blue⁻CD14⁻CD20⁺IgG⁺CD27⁺IgM⁻gp140-F^hi, while CD4bs-specific memory B cells were defined as CD3⁻CD8⁻Aqua Blue⁻CD14⁻CD20⁺IgG⁺CD27⁺IgM⁻gp140-F^higp140-F–D368R^lo.

Wang Y., Sundling C., Wilson R., O’Dell S., Chen Y., Dai K., Phad G.E., Zhu J., Xiao Y., Mascola J.R., Karlsson Hedestam G.B., Wyatt R.T, & Li Y. (2016). High Resolution Longitudinal Study of HIV-1 Env Vaccine-elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence. Journal of immunology (Baltimore, Md. : 1950), 196(9), 3729-3743.

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Flow Cytometry Cell Purity Assay

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Primary cells were stained for purity post-isolation using anti-CD45 (HI30, BD), anti-CD8 (RPA-T8, BD), and Near IR Live/dead stain (Invitrogen), then fixed with 4% formaldehyde (Thermo) and analyzed using a BD LSR II or a Cytek Aurora cytometer. Data were analyzed using FlowJo 10 software.

Do P., Perdue L.A., Chyong A., Hunter R., Dougan J., Henry C.J., Porter C.C, & Dreaden E.C. (2020). Rapid Assembly and Screening of Multivalent Immune Cell-Redirecting Therapies for Leukemia. ACS combinatorial science, 22(10), 533-541.

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Gating Lymphocytes and Monocytes for Flow Cytometry

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Live lymphocytes and monocytes were gated by forward and side scatter. T cells were identified by anti–CD3-FITC (Biolegend HIT3a, all patients), CD8-APC (BD RPA-T8; Pts 1 and 2), and CD4-BV650 (BD L200; Pts 1 and 2). Monocytes were identified by anti–CD14-APC/Cy7 (BioLegend M5E2; pts 1 and 2) or CD14-APC (BD M5E2; Pts 3, 6 and 7). A healthy PBMC donor was used as control for all flow cytometry experiments.

Liu M., Tayob N., Penter L., Sellars M., Tarren A., Chea V., Carulli I., Huang T., Li S., Cheng S.C., Le P., Frackiewicz L., Fasse J., Qi C., Liu J.F., Stover E.H., Curtis J., Livak K.J., Neuberg D., Zhang G., Matulonis U.A., Wu C.J., Keskin D.B, & Konstantinopoulos P.A. (2022). Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer. Clinical Cancer Research, 28(15), 3356-3366.

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Plasma Cell Differentiation Assay

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B cells obtained from human PBMCs (described above) were seeded in 96-well plates at 2.5 × 10⁶ cells/mL, then cultured at 37°C with 5% CO₂ for 3 days in RPMI supplemented with 10% FBS, 100 U/mL Pen/Strep, 0.11 mg/mL sodium pyruvate, 10 mM HEPES, 2 mM glutamine, 55 μM 2ME with IL-21 (50 ng/mL), anti-CD40 (5 μg/mL, IC10 Southern Biotech), and R848 (Resiquimod, 3 μg/mL, Sigma-Aldrich). CAR T cells were added to B cells at a 1:1 cell ratio, keeping the total cell concentration between 0.5–1 × 10⁶ in B cell media as above, but with the addition of IL-2 (50 ng/mL), IL-7 (5 ng/mL), and IL-15 (5 ng/mL). The relative proportion of CD4⁻CD8⁻ BCMA⁺ plasma cells in each sample was then assessed 2 days post-addition of T cells by flow cytometry after staining with FITC anti-CD4 (RPA-T4, BD Bioscience), PerCP-Cy5.5 anti CD-8 (RPA-T8, BD Bioscience), PE anti-BCMA (19F2, BioLegend), Alexa700 anti-CD138 (MI15, Biolegend), and PE-Cy7 anti-CD19 (HIB19, eBioscience).

Hale M., Lee B., Honaker Y., Leung W.H., Grier A.E., Jacobs H.M., Sommer K., Sahni J., Jackson S.W., Scharenberg A.M., Astrakhan A, & Rawlings D.J. (2017). Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells. Molecular Therapy. Methods & Clinical Development, 4, 192-203.

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