Clone rpa t4

Manufactured by BD

Sourced in United States

The Clone RPA-T4 is a laboratory instrument designed for automated liquid handling tasks. It utilizes Robotic Pipetting Arms (RPA) technology to perform precise and consistent liquid transfers. The core function of the Clone RPA-T4 is to automate various liquid handling protocols, enabling efficient and reproducible sample processing in a laboratory setting.

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RPA-T4 (62 citations) CD4 (clone RPA-T4), (13 citations) Anti-CD4 (clone RPA-T4; (5 citations) CD4-APC-H7 (clone RPA-T4, (5 citations) CD4-V450 (clone RPA-T4; (5 citations) Anti-CD4-FITC (clone RPA-T4) (5 citations) CD4APC (clone RPA-T4, (5 citations) CD4 v500 (clone RPA-T4, (4 citations) CD4-APC-Cy7 (clone RPA-T4), (4 citations) Anti-CD4-PE (clone RPA-T4), (3 citations)

11 protocols using clone rpa t4

Expansion and Immunophenotyping of T Cells

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To expand activated T cells, PBMCs were added to flasks pre-coated with anti-CD3 (1:500, Clone OKT-3, Invitrogen) and anti-CD28 (1:500, Clone CD28.2, Invitrogen) monoclonal antibodies and then incubated in serum-free KBM551 medium (Kohjin Bio) containing 200 IU/ml human recombinant interleukin-2 (rhIL-2) (R&D Systems) at 37 °C in a 5% CO₂ atmosphere. Four days later, the cells were transferred into a culture bag (Kohjin Bio) and were cultured for a total of 10–14 days. Cultured T cells were harvested and analyzed by flow cytometry. The following monoclonal antibodies were used to stain cultured T cells: anti-CD45-BUV395 (1:200, Clone HI30, BD Biosciences), anti-CD3-PE-Cy7 (1:100, Clone SP34-2, BD Biosciences), anti-CD56-PE (1:100, Clone CMSSB, Invitrogen), anti-CD19-PE-CF594 (1:200, Clone HIB19, BD Biosciences), anti-CD14-PE-CF594 (1:200, Clone MQP9, BD Biosciences), anti-CD4-BV605 (1:100, Clone RPA-T4, BD Biosciences), anti-CD8-APC-R700 (1:100, Clone SK1, BD Biosciences), anti-CCR7-PerCP-Cy5.5 (1:100, Clone G043H7, Biolegend), and anti-CD45RA-APC-H7 (1:100, Clone HI100, BD Biosciences).

Kim A., Lee K.G., Kwon Y., Lee K.I., Yang H.M., Habib O., Kim J., Kim S.T., Kim S.J., Kim J.S, & Hwang D.Y. (2021). Off-the-Shelf, Immune-Compatible Human Embryonic Stem Cells Generated Via CRISPR-Mediated Genome Editing. Stem Cell Reviews and Reports, 17(3), 1053-1067.

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Activated T Cell Phenotyping by Flow Cytometry

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Flow cytometry was performed on a FACS LSR II or Fortessa (BD Biosciences). For lymphocyte activation studies, soluble anti-CD3 (500 ng/ml; clone OKT3, Bio X Cell) ± anti-CD28 (1 μg/ml; clone CD28.2, BD Biosciences) or IL-2 (PeproTech) was added to media for 2 days. Proliferation was measured by CFSE (eFluor 670, BD Biosciences) after 3 days. EVs were added every 24 hours (5 μg/ml). PD1 blockade was achieved by adding anti-PD1 (10 μg/ml; EH12) or immunoglobulin G control (clone MOPC-21, Bio X Cell). Cells were gated by FSC (forward scatter)/SCC (side scatter) while excluding duplets by FSC-A/FSC-H. Subsequently, CD3⁺CD56⁻ T cells were gated with further classification of CD4⁺ and CD8⁺ T cells. Finally, CD4⁺ and CD8⁺ T cells were measured for their CD69, CD25, PD1, and TIM3 expression (seen as a downloadable figure). Cells were stained with the following antibodies: CD3/PE-Cy5.5 (1:100; clone SK7, eBioscience), CD4/FITC (1:100; clone RPA-T4, BD Biosciences), CD8/AmCyan (1:10; clone SK1, BD Biosciences), CD16/APC-Cy7 (1:100, BD Biosciences), CD25/PE-Cy7 (1:10; clone BC96, eBioscience), CD56/V450 (1:30; clone B159, BD Biosciences), CD69/APC (1:10; clone FN50, BD Biosciences), TIM3/APC-Cy7 (1:100, clone F38-2E2, BD Biosciences), and PD1/PE (1:100; clone J105, eBioscience).

Ricklefs F.L., Alayo Q., Krenzlin H., Mahmoud A.B., Speranza M.C., Nakashima H., Hayes J.L., Lee K., Balaj L., Passaro C., Rooj A.K., Krasemann S., Carter B.S., Chen C.C., Steed T., Treiber J., Rodig S., Yang K., Nakano I., Lee H., Weissleder R., Breakefield X.O., Godlewski J., Westphal M., Lamszus K., Freeman G.J., Bronisz A., Lawler S.E, & Chiocca E.A. (2018). Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Science Advances, 4(3), eaar2766.

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Modulation of T cell proliferation by granulocytes

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Sorted CD3⁺ T cells were labeled with 10 μmol/L carboxy-fluorescein diacetate succinimidyl ester (CFSE; Life Technologies) and cultured with sorted granulocytes at ratios of 1:0, 1:0.5, 1:1 in complete media at 37°C, 5% CO₂ for 4 days in the presence of 1:1 ratio of anti-CD3/ anti-CD28 dynabeads (Invitrogen). Cells were stained with V450 anti-CD4 (Clone-RPA-T4; BD Biosciences) and APC-Cy7 anti-CD8 (Clone-RPA-T8; BioLegend) and proliferation was determined by CFSE dilution. Unstimulated T cells were used as a negative control. The effect of the addition of L-NMMA (0.5 mmol/L, NG-Methyl-L-arginine acetate), nor-NOHA (0.5 mmol/L, N-Omega-hydroxy-nor-L-arginine) and iNAC (10 mmol/L; all from Sigma- Aldrich) was similarly tested. The percentage of cells that diluted CFSE (divided cells) was determined.

Khanna S., Graef S., Mussai F., Thomas A., Wali N., Yenidunya B.G., Yuan C., Morrow B., Zhang J., Korangy F., Greten T.F., Steinberg S.M., Stetler-Stevenson M., Middleton G., De Santo C, & Hassan R. (2018). Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(12), 2859-2872.

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Adipose Tissue Immune Cell Isolation

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AT from dermolipectomies was digested using type I collagenase (Sigma-Aldrich). Whole AT was sequentially digested with 1:1 dispase (2.4 U/mL in phosphate-buffered saline (PBS), Gibco, 30 min at 37 °C with shaking) and type I collagenase (250 U/mL in PBS 2% bovine serum albumin (BSA), Sigma-Aldrich, 30 min at 37 °C with shaking). After digestion, the cell suspension was filtered through a 250-µm strainer and centrifuged. The erythrocyte lysis step was performed followed by successive filtrations through 100, 70, and 40 -µm strainers. The viable recovered cells were counted and further analyzed by flow cytometry. Immune cells were enriched using a CD45 isolation kit (Stem Cells Technologies) following the manufacturer’s protocol. Anti-human FITC-CD4 dilution (1/10), BD Pharmingen, Ref 555346, CLONE RPA-T4 (RUO); PerCP-CD8 dilution (1/10), BD, Ref 345774, CLONE SK1 (CE/IVD); Pe-Cy7-CD56 dilution (1/20); BD Pharmingen, Ref 345774, CLONE B159 (RUO); APC-Cy7-CD19 dilution (1/20); BD Pharmingen, Ref 557791, CLONE SJ25C1 (RUO); APC-CD25 dilution (1/20), BD, ref 340907, CLONE 2A3 (CE/IVD); V450-CD3 diution (1/20); BD Horizon, ref 560365, CLONE UCHT1 (RUO); BV510-CD45 dilution (1/20), BIOLEGEND, ref 304036, CLONE HI30.Flow cytometry was carried out with LSRII BD Fortessa. Cells are expressed as number/g adipose tissue.

Zamora A., Nougué M., Verdu L., Balzan E., Draia-Nicolau T., Benuzzi E., Pujol F., Baillif V., Lacazette E., Morfoisse F., Galitzky J., Bouloumié A., Dubourdeau M., Chaput B., Fazilleau N., Malloizel-Delaunay J., Bura-Rivière A., Prats A.C, & Garmy-Susini B. (2024). 15-Lipoxygenase promotes resolution of inflammation in lymphedema by controlling Treg cell function through IFN-β. Nature Communications, 15, 221.

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Phenotypic Analysis of DC and CIK Cells

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DC and CIK cells cultured on day 1 and day 7 were collected. After washing with PBS and centrifugation, the DC cells were incubated with FITC-conjugated mouse anti-human CD14 (Clone M5E2, Cat. No. 561712, BD Pharmingen, US), CD83 (Clone HB15e, Cat. No. 560929, BD Pharmingen, US) and CD86 (Clone 2331 (FUN-1), Cat. No. 560958, BD Pharmingen, US) monoclonal antibodies and PE-labeled mouse anti-human HLA-DR monoclonal antibody (Clone G46–6, Cat. No. 560943, BD Pharmingen, US) for 20 min at room temperature. The CIK cells were incubated with FITC-conjugated mouse anti-human CD3 (Clone HIT3a, Cat. No. 561802, BD Pharmingen, US) and CD4 (Clone RPA-T4, Cat. No. 561005, BD Pharmingen, US) monoclonal antibodies and PE-labeled mouse anti-human CD8 (Clone RPA-T8, Cat. No. 561949 BD Pharmingen, US) and CD56 (Clone B159, Cat. No. 561903, BD Pharmingen, US) monoclonal antibodies for 20 min at room temperature. Then the DC and CIK cells were washed with PBS twice. Flow cytometry was used to determine the phenotypes of DC and CIK cells.

Yang T., Zhang W., Wang L., Xiao C., Wang L., Gong Y., Huang D., Guo B., Li Q., Xiang Y, & Nan Y. (2018). Co-culture of dendritic cells and cytokine-induced killer cells effectively suppresses liver cancer stem cell growth by inhibiting pathways in the immune system. BMC Cancer, 18, 984.

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Multiparametric Flow Cytometry

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Phycoerythrin (PE)-conjugated anti-Bcl-2 (Clone Ms IgG1), fluorescein (FITC)-conjugated anti-CD25 (Clone M-A251), PE-conjugated anti-CD132 (Clone AG184), allophycocyanin (APC; Clone RPA-T4) and Peridinin-chlorophyll proteins (PerCP; Clone RPA-T4)-conjugated anti-CD4, PerCp (Clone SK1) or FITC-conjugated anti-CD8 (Clone HIT8a), Alexa Fluor 647-conjugated (Clone HIL-7R-M21) anti-CD127 or PE (Clone HIL-7R-M21), as well as PE-conjugated anti-phosphorylated STAT5 (p-STAT5; Clone pY694) were purchased from BD Pharmingen (San Diego, CA, USA).

Albareda M.C., Perez-Mazliah D., Natale M.A., Castro-Eiro M., Alvarez M.G., Viotti R., Bertocchi G., Lococo B., Tarleton R.L, & Laucella S.A. (2015). Perturbed T cell IL7 receptor-signaling in chronic Chagas disease. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3883-3889.

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Proliferation Assay for T and B Cells

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PBMCs, either freshly isolated or thawed from liquid nitrogen−stored samples, were incubated with CellTrace violet Cell Proliferation Kit (1μM; Invitrogen). After 20 minutes, 10 volumes of RPMI/10% fetal bovine serum (FBS) were added, and the cells were washed twice with RPMI/10% FBS. 1 × 10⁵ cells were seeded into 96-well plates, stimulated with anti-CD3 antibody or anti-CD28 antibody (1 μg/mL each, eBioscience; 16–0037-85 and 16–0289-85). For B cell proliferation, cells were stimulated with anti-IgM (10 μg/ml, Jackson Lab; 109–006-129), CD40L (100 ng/ml, Enzo; ALX522–110-C010), CpG (0.5 μM, Enzo; ALX 746–006-C100) IL-4 (50 ng/ml, Peprotech; 200–04), and IL-21 (50ng/ml, Peprotech; 200–21) as indicated in the figure. After incubation for 3–5 days (3 days for T cells and 4–5 days for B cells), cells were stained with fluorochrome-conjugated CD4, CD8, or CD19 (BD Biosciences, Clone RPA-T4, RPA-T8, HIB19 respectively) for 30 minutes at 4°C (dark). Cells were washed with PBS twice, and cells were acquired and analyzed by flow cytometry (Becton Dickinson FACSCanto II) and FlowJo software (FlowJo 10.5.2, TreeStar).

Kuehn H.S., Bernasconi A., Niemela J.E., Almejun M.B., Gallego W.A., Goel S., Stoddard J.L., Sánchez R.G., Franco C.A., Oleastro M., Grunebaum E., Ballas Z., Cunningham-Rundles C., Fleisher T.A., Franco J.L., Danielian S, & Rosenzweig S.D. (2020). A Nonsense N –Terminus NFKB2 Mutation Leading to Haploinsufficiency in a Patient with a Predominantly Antibody Deficiency. Journal of Clinical Immunology, 40(8), 1093-1101.

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Isolation and Stimulation of Tfh Cells

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CD4+ T cells were isolated from previously frozen PBMC by negative selection using CD4+ T cell isolation kit II per the manufacturers instructions (Miltenyi, San Diego, CA). The isolated CD4 cell population was stained as described above. Tfh cells (CD4+CD3+CD45RO+CXCR5+) were isolated using a BD FACSAria II cell sorter. After sorting, cells were plated in a round-bottom 96-well plate at 2 × 10⁵/well in 200 μl serum-free AIM-V medium (Life Technologies) with human rIL-7 (4 ng/ml). Tfh cells were incubated in medium alone, with TCR stimulation (Dynabeads, human Tactivator CD3/28, Life technologies) or with human rIL-2 (125 μg/ml), all in the presence or absence of anti-human IL-2 (50 μg/ml). After 5 days of incubation, cells were harvested, washed and surface stained with antibodies for CD4 (eBioscience, San Diego, CA, clone RPA-T4), CD3 (BD, Franklin Lakes, NJ, clone UCHT1), CD45RO (eBioscience, clone UCHL1), CXCR5 (Biolegend, clone J252D4). For exclusion, stains for CD19 (BD, clone HIB19), CD14 (BD, clone M5E2), CD8 (BD, clone RPA-T8) were also performed. Intracellular stains for FOXP3 and Helios were performed as described above.

Schulten V., Tripple V., Seumois G., Qian Y., Scheuermann R.H., Fu Z., Locci M., Rosales S., Vijayanand P., Sette A., Alam R., Crotty S, & Peters B. (2017). Allergen-specific immunotherapy modulates the balance of circulating Tfh and Tfr cells. The Journal of allergy and clinical immunology, 141(2), 775-777.e6.

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Immune Cell Polarization and Sorting

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For in vitro polarization assays, we used recombinant human IL-12 [20ng/mL], IL-4 [20ng/mL], IL-23 [10ng/mL], IL-6 [20ng/mL], TGF-β1 [10ng/mL] and IL-1β [10ng/mL] cytokines (all purchased from R&D Systems) and neutralizing human IFN-γ [10g/mL], IL-4 [10g/mL], IL-23p19 [10g/mL], IL-6 [10g/mL], TGF-β [10g/mL], and IL-1 [10g/mL] antibodies (all purchased from R&D Systems). For sorting of live gut-resident CD4 T cells used in ATAC-Seq experiments, we stained cells with Sytox-Blue Dead Cell Stain (Invitrogen – Cat. Number S34857), APC anti-human CD3 (BD Biosciences – Cat. Number 555335), FITC anti-human CD4 (Biolegend – Clone RPA-T4) and PE anti-human CD8 (BD Biosciences – Cat. Number 555635) antibodies. For single cell RNA-Seq experiments, we used the same antibodies described for ATAC along with APC/Cy7 anti-human CD161 (Biolegend – Clone HP-3G10) and PerCP/Cy5.5 anti-human TCR V24-J18 (Biolegend – Clone 6B11) antibodies.

Medina T.S., Murison A., Smith M., Kinker G.S., Chakravarthy A., Vitiello G.A., Turpin W., Shen S.Y., Yau H.L., Sarmento O.F., Faubion W., Lupien M., Silverberg M.S., Arrowsmith C.H, & De Carvalho D.D. (2023). The chromatin and single-cell transcriptional landscapes of CD4 T cells in inflammatory bowel disease link risk loci with a proinflammatory Th17 cell population. Frontiers in Immunology, 14, 1161901.

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Flow Cytometric Analyses of Leukocyte Surface Markers

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Surface markers of whole-blood leukocytes were determined by standard flow cytometric analyses using FACS Calibur and Cellquest software (BD).¹ Leukocytes were gated into lymphocytes, monocytes, and granulocytes by forward- and side-scatter analysis. Percent-positive cells were quantified via direct immunofluorescence staining using fluorescein isothiocyanate (FITC)-conjugated antibodies with phycoerythrin (PE)-conjugated antibodies. After binding of fluorescently labeled antibodies, expression densities of individual antigens were recorded. The expression density of the relevant antigens was calculated as the mean fluorescence intensity (MFI) according to the equation:
Subsequent monoclonal antibodies were determined separately on monocytes: antibodies directed against HLA-DR (clone L243, BD), CD83 (clone HB15a, Beckman Coulter), and CD123 (clone 9F5, BD). Lymphocytes were evaluated using antibodies directed against CD25 (clone M-A251, BD Biosciences) on its own and together with CD4 (cloneRPA-T4, BD Biosciences), CD2 (clone 39C1.5, Beckman Coulter) either together with CD80 (clone L307.4, Immunotech) or CD86 (clone B-T7; Diaclone). A mouse FITC-IgG1 antibody (clone X40) in conjunction with PE-conjugated IgG2a (clone X39, both from BD Biosciences) served as the isotype controls.

Bizjak D.A., Treff G., Zügel M., Schumann U., Winkert K., Schneider M., Abendroth D, & Steinacker J.M. (2021). Differences in Immune Response During Competition and Preparation Phase in Elite Rowers. Frontiers in Physiology, 12, 803863.

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