Anti cd3 clone sk7

Manufactured by BD

Sourced in Germany

Anti-CD3 (clone SK7) is a monoclonal antibody that recognizes the CD3 complex on human T cells. The CD3 complex is essential for T cell activation and signal transduction. This antibody can be used for the identification and enumeration of T cells in flow cytometry applications.

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8 protocols using anti cd3 clone sk7

Characterization of CD8 T cells and moDC's

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CD8 T cells were harvested and incubated with HLA-multimers for 15 min at 4 °C and washed three times with cold PBS/BSA prior to staining with antibodies. Cells were incubated with anti-CD3 (clone SK-7, BD), anti-CD4 (clone SK-3, BD), anti-CD8 (clone SK-1, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. T cell activation was measured by intracellular IFNγ staining (XMF1.2, BioLegend) using an intracellular cytokine staining kit (BioLegend) according to manufactures protocol. moDC’s were stained with anti-CD1a (clone HI149, BD), anti-CD14 (clone M5E2, BD), anti-CD80 (clone L307.4, BD), anti-CD83 (clone HB15e, BD), anti-CD86 (clone IT2.2, BioLegend), and anti-HLA-DR (clone G46-6, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. Samples were acquired using a BD LSRFortessa™ flow cytometry machine and analyzed using FlowJo software (Tree Star).

Marijt K.A., Griffioen L., Blijleven L., van der Burg S.H, & van Hall T. (2021). Cross-presentation of a TAP-independent signal peptide induces CD8 T immunity to escaped cancers but necessitates anchor replacement. Cancer Immunology, Immunotherapy, 71(2), 289-300.

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Flow Cytometric Analysis of Immune Cells and Tumors

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For analysis of purity, 2 × 10⁵ Vγ9Vδ2 γδ T cells were washed and stained with mAb as follows: anti-CD3 (clone SK7, BD Biosciences, Heidelberg, Germany), anti-TCRγδ (clone 11F2, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-TCRαβ (clone IP26, BioLegend), anti-TCRVδ2 (clone Immu389, Beckman Coulter, Krefeld, Germany), anti-TCRVγ9 (clone 7A5 [56 (link)]), and corresponding isotype controls (BD Biosciences or BioLegend) for 25 min. After washing, cells were analyzed by flow cytometry (LSR-Fortessa, BD Biosciences) using Diva 8 or FlowJo software.
For surface staining of PDAC cells, 2 × 10⁵ cells were washed and treated with 20 µL of a 1:20 diluted Fc-blocking reagent (Miltenyi Biotec) for 15 min. After a washing step, cells were stained with PE-Vio770-conjugated anti-HER-2 clone 24D2 mAb (Miltenyi Biotec) or appropriate isotype control and analyzed on a flow cytometer. For intracellular staining, 2 × 10⁵ tumor cells were permeabilized and fixed with Cytofix/Cytoperm kit (BD Biosciences) and stained with 10 μg/mL anti-pan-IDO-APC mAb (clone #700838, R&D Systems, Wiesbaden, Germany) or corresponding isotype controls. After washing, all samples were analyzed by flow cytometer (FACS Calibur Analyzer, BD Biosciences) using CellQuestPro or FlowJo software.

Jonescheit H., Oberg H.H., Gonnermann D., Hermes M., Sulaj V., Peters C., Kabelitz D, & Wesch D. (2020). Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on γδ T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells. Cells, 9(5), 1140.

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Cytotoxic T-cell Activation Assay

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Cryopreserved PBMCs were thawed and stabilized overnight at 37°C. The cells were pre-incubated for 30 minutes at 37°C, 5% CO₂ with Polymyxin B (Sigma Aldrich) at a concentration of 10 μg/mL. The cells were then incubated for 16 hours with 100 μM of each recombinant protein or peptide in the presence of 2 μg/ml of anti-human CD28 (clone CD28.2, BD Biosciences) and anti-CD49d (clone 9F10, BioLegend) antibodies and IL-2 (50 IU/ml, Peprotech, Cranbury, NJ) and IL-7 (5 ng/ml, Peprotech). To detect intracellular staining, eBioscience™ Protein Transport Inhibitor Cocktail (500X, ThermoFisher Scientific) was added during the final 5 hours of culture. After 16 hours, the cells were labeled with Fixable Viability Stain 510 (BD Biosciences) for live cell staining and fluorophore conjugated anti-CD3 (clone SK7, BD Biosciences), CD4 (clone RPA-T4, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), Granzyme B (clone GB11, BD Biosciences), IFNγ (clone B27, BD Biosciences) and IL-17A (clone BL168, BD Biosciences) antibodies and detected using a BD LSR Fortessa.

Lanz T.V., Brewer R.C., Ho P.P., Moon J.S., Jude K.M., Fernandez D., Fernandes R.A., Gomez A.M., Nadj G.S., Bartley C.M., Schubert R.D., Hawes I.A., Vazquez S.E., Iyer M., Zuchero J.B., Teegen B., Dunn J.E., Lock C.B., Kipp L.B., Cotham V.C., Ueberheide B.M., Aftab B.T., Anderson M.S., DeRisi J.L., Wilson M.R., Bashford-Rogers R.J., Platten M., Garcia K.C., Steinman L, & Robinson W.H. (2022). Clonally Expanded B Cells in Multiple Sclerosis Bind EBV EBNA1 and GlialCAM. Nature, 603(7900), 321-327.

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FACS Analysis of T-cell Subsets

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Fluorescence-activated cell sorting (FACS) was performed using the FACS Aria Fusion flow cytometer (BD, USA). PBMC and CBMC were incubated with 5% Fc-receptor block before staining. The following antibodies were used: anti-CD3 (clone REA613; Miltenyi Biotec), anti-CD3 (clone SK7; BD Bioscience), anti-γδ TCR (clone 11F2, BD Bioscience or Miltenyi Biotec), anti-Vγ9 (clone IMMU 360; Beckman Coulter), anti-Vδ2 (clone 123R3; Miltenyi Biotec).

Deng L., Harms A., Ravens S., Prinz I, & Tan L. (2022). Systematic pattern analyses of Vδ2+ TCRs reveal that shared “public” Vδ2+ γδ T cell clones are a consequence of rearrangement bias and a higher expansion status. Frontiers in Immunology, 13, 960920.

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Flow Cytometry Analysis of Regulatory T Cells

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All clinical flow cytometry (FACS) was performed following good clinical practice at the Department of Clinical Immunology, Addenbrooke’s Hospital, Cambridge, UK, within 4 h of phlebotomy. Operators were blinded to the aldesleukin dose allocated. A 2.6-ml sample of peripheral whole blood was collected into EDTA tubes, and 50 μl was stained with specific fluorochrome-conjugated antibodies at room temperature for 15 min to identify Tregs as CD3⁺CD4⁺CD25^highCD127^low T cells. The clones used were anti-CD3 (clone SK7, phycoerythrin [PE]-Cy7-labelled; BD Biosciences), anti-CD4 (clone RPA-T4, FITC-labelled; BD Biosciences), anti-CD127 (clone HIL-7R-M21, PE-labelled; BD Biosciences), anti-CD25 (clone M-A251 and 2A3, allophycocyanin [APC]-labelled; BD Biosciences), anti-CD45RA (clone HI100, APC-Cy7-labelled; BioLegend), and anti-CD62L (clone DREG-56, PerCP/Cy5.5-labelled; BioLegend). Red cells were then lysed (BD FACS Lysing Solution); the cells were washed and resuspended in BD Cell Fix and then immediately analysed on a BD FACSCanto II flow cytometer utilising FACSDiva software (BD Biosciences). In parallel, a whole-blood BD Multitest 6-Color TBNK assay using BD Trucount Tubes according to the manufacturers’ instructions (BD Biosciences) was run to determine the relative and absolute concentration of lymphocyte subpopulations, including T, B, and NK cells.

Todd J.A., Evangelou M., Cutler A.J., Pekalski M.L., Walker N.M., Stevens H.E., Porter L., Smyth D.J., Rainbow D.B., Ferreira R.C., Esposito L., Hunter K.M., Loudon K., Irons K., Yang J.H., Bell C.J., Schuilenburg H., Heywood J., Challis B., Neupane S., Clarke P., Coleman G., Dawson S., Goymer D., Anselmiova K., Kennet J., Brown J., Caddy S.L., Lu J., Greatorex J., Goodfellow I., Wallace C., Tree T.I., Evans M., Mander A.P., Bond S., Wicker L.S, & Waldron-Lynch F. (2016). Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial. PLoS Medicine, 13(10), e1002139.

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Flow Cytometric Analysis of T Cell Activation

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T cells (3×10⁶) and tetramers (0.2 μg per 50 μl staining volume) were incubated for 10 min at room temperature in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA). Subsequently anti-CD3 (clone SK7, BD Biosciences) antibody was added an incubated for 10 min at room temperature. Subsequently, additional antibodies (anti-TRBV4–1, IM2287, Beckman Coulter) were added and incubated for 20 min at 4°C. Cells were washed with PBS with 1% BSA and analyzed on an LSRFortessa (BD Biosciences) flow cytometer. For display in a matrix, percentage positive cell or mean fluorescence intensity of the positive cells were normalized to Z score and a heat map was created using the heatmap.2 function of R (https://cran.r-project.org/web/packages/gplots/index.html).

Reinink P., Shahine A., Gras S., Cheng T.Y., Farquhar R., Lopez K., Suliman S.A., Reijneveld J.F., Le Nours J., Tan L.L., León S.R., Jimenez J., Calderon R., Lecca L., Murray M.B., Rossjohn J., Moody D.B, & Van Rhijn I. (2019). A T cell receptor β chain motif biases towards recognition of human CD1 proteins. Journal of immunology (Baltimore, Md. : 1950), 203(12), 3395-3406.

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Isolation and Expansion of CD3/CD1c-Reactive T Cells

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PBMCs from subject HD1 were sorted based on their binding to anti-CD3 (clone SK7, Becton Dickinson) and CD1c tetramers treated with a mixture of MAG and fatty acid. Population expansion of sorted cells was performed using anti-CD3 (clone OKT3, produced in-house), irradiated feeder cells and IL-2. After 2 weeks, the sorting and expansion procedure was repeated, and the resulting cell line was named ‘HD1’. IFN-γ ELISpot assays were performed using anti-1D1K and GB-11-biotin according to the manufacturer’s instructions (Mabtech). After an initial screen using V_β-specific antibodies (V_β2: IM1484; V_β4: IM3602; V_β5.1: IM1552; V_β9: IM2003; V_β13.6: IM1330 and V_β7.1: IM2287 (Beckman Coulter), Vβ13.1 (clone H31, Ebioscience), Vβ8 (clone JR2, Biolegend), V_β21.3 (Catalog: 1483, Immunotech), three subsets of HD1 were sorted using anti-Vβ5.1 and cultured in vitro. The TCR β-chain sequences of HD1CD4⁺ and HD1CD4^– were determined from RNA isolated with an RNeasy Kit (Qiagen), with cDNA synthesized using a Quantitect Reverse Transcription Kit (Qiagen). V-segment usage was determined by PCR using primer set IPS000030, as described online (https://www.imgt.org/) and via a multiplex approach⁴⁶.

Wun K.S., Reijneveld J.F., Cheng T.Y., Ladell K., Uldrich A.P., Le Nours J., Miners K.L., McLaren J.E., Grant E.J., Haigh O.L., Watkins T.S., Suliman S., Iwany S., Jimenez J., Calderon R., Tamara K.L., Leon S.R., Murray M.B., Mayfield J.A., Altman J.D., Purcell A.W., Miles J.J., Godfrey D.I., Gras S., Price D.A., Van Rhijn I., Moody D.B, & Rossjohn J. (2018). T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids. Nature immunology, 19(4), 397-406.

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Quantifying Immune Cell Populations in Tissue

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Sections (5 μm each) were cut and mounted on StarFrost adhesive glass slides (Knittelgläser, Braunschweig, Germany). Sealed slides were stored at −80°C until further use. LN tissue sections were stained using mouse monoclonal antibodies against T cells (anti-CD3, clone SK7; Becton Dickinson, Breda, the Netherlands), B cells (anti-CD22, clone RFB4; Millipore, Amsterdam, the Netherlands) and plasma cells (anti-CD138, clone MI15; Dako, Heverlee, Belgium).
Staining was performed using a three-step immunoperoxidase method to detect bound anti-CD138 antibodies, as described previously [17 (link)]. For anti-CD3 and anti-CD22, we used a two-step immunoperoxidase method with a secondary polymer—horseradish peroxidase—conjugated anti-mouse antibody (EnVision+ System; Dako). As a negative control, irrelevant isotype-matched immunoglobulins, instead of the primary antibody, were applied to the sections. Staining was analysed by digital image analysis in a blinded fashion using a Syndia algorithm on a Qwin-based analysis system (Leica, Cambridge, UK) as described previously [18 (link)]. The number of positive cells was calculated for each section as the number of positive cells per square millimetre of tissue.

Ramwadhdoebe T.H., van Baarsen L.G., Boumans M.J., Bruijnen S.T., Safy M., Berger F.H., Semmelink J.F., van der Laken C.J., Gerlag D.M., Thurlings R.M, & Tak P.P. (2019). Effect of rituximab treatment on T and B cell subsets in lymph node biopsies of patients with rheumatoid arthritis. Rheumatology (Oxford, England), 58(6), 1075-1085.

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