Cd3 sk7

Manufactured by BD

Sourced in United States, Germany

The CD3 (SK7) is a cell surface glycoprotein that serves as a co-receptor during antigen recognition and T-cell activation. It is expressed on mature T cells and plays a critical role in the initiation of the T-cell activation cascade.

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Lab products found in correlation

Cd8 sk1, by BD (7 mentions) Cd19 sj25c1, by BD (7 mentions) Igd ia6 2, by BD (4 mentions) Igm mhm 88, by BioLegend (4 mentions) Cd4 sk3, by BD (3 mentions) Cd45ra hi100, by BD (3 mentions) Foxp3 pch101, by Thermo Fisher Scientific (3 mentions) Facscanto 2 flow cytometer, by BD (3 mentions) Cd11c b ly6, by BD (2 mentions) Cd20 l27, by BD (2 mentions) Cd16 3g8, by BD (2 mentions) Live dead violet, by Thermo Fisher Scientific (2 mentions) Cd45 hi30, by BD (2 mentions) Il 7rα a019d5, by Thermo Fisher Scientific (2 mentions) Cd11c bu15, by Abcam (2 mentions) Cytofix cytoperm, by BD (2 mentions) Live dead blue, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) Brefeldin a, by Merck Group (2 mentions) Il 2 mq1 17h12, by BioLegend (2 mentions)

Spelling variants of this product

Anti-CD3 (clone SK7, (8 citations) Human anti-CD3 antibody (clone SK7, (3 citations) CD3-FITC (SK7) (3 citations) CD3-APC Cy7 (SK7; (3 citations) FITC–anti-human CD3 (clone SK7) (2 citations) CD3 (SK7)-AF700 (2 citations) CD3 AmCyam (clone SK7, (2 citations) APC‐H7‐conjugated anti‐human CD3 antibody (clone SK7; (2 citations) Anti-human CD3-APC-H7 (clone SK7; (2 citations) Anti-CD3-APC (SK7) (2 citations)

19 protocols using cd3 sk7

Comprehensive NK Cell Phenotyping

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dNK cells were gated on as live, CD9⁺CD56⁺ cells. pbNK cells were gated on as live CD56⁺CD3⁻ cells. The following Abs were used: Live/Dead discriminator (Life Technologies), CD9 (SN4 or M-L13 from eBioscience or BD Biosciences, respectively), CD56 (HCD56 from BioLegend), and CD3 (SK7) from BD Biosciences. Fibroblasts and macrophages were identified using CD10 (HI10a from BioLegend) and CD14 Abs (MφP9 and HCD14 from BD Pharmingen and BioLegend), respectively. The following Abs were used to stain KIRs: UPR1 (KIR2DL5) from BioLegend and Carlos Vilches (33 (link)); 179315 (KIR2DS4), 143211 (KIR2DL1), and 181703 (KIR2DL4) from R&D Systems; FES172 (KIR2DS4) and EB6 (KIR2DL1/S1) from Beckman Coulter; CHL (KIR2DL2/3/S2) from BD Pharmingen; DX9 (KIR3DL1) from BioLegend; NKVFS1 (KIR2DL1/2/3/S1/2/4) from Abcam; 5.133 (KIR3DL2) from Marco Colonna (34 (link)); and FLAG Abs from Sigma-Aldrich. Intracellular staining was performed according to the manufacturers’ instructions with Abs against Ki647 (BD Pharmingen), CCL3 (R&D Systems), and GM-CSF (BD Biosciences).

Kennedy P.R., Chazara O., Gardner L., Ivarsson M.A., Farrell L.E., Xiong S., Hiby S.E., Colucci F., Sharkey A.M, & Moffett A. (2016). Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy. The Journal of Immunology Author Choice, 197(11), 4292-4300.

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Multiparametric Immunofluorescence Profiling

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Frozen skin sections were fixed with acetone and blocked with 10% normal goat serum (Vector Laboratories) for 30 minutes. Primary antibodies were incubated overnight at 4°C and amplified with the appropriate Alexa Fluor® 488 (A-488) or 568 (A-568) conjugated secondary antibody for 30 minutes at room temperature. Antibodies used are: IL-32αβγδ (KU32–52, BioLegend), CD3 (SK7, BD Biosciences), CD4 (SK3, BD Biosciences), CD8 (SK1, BD Biosciences), hNKp46 (195314, R&D Systems), CD20 (L27, BD Biosciences), CD14 (M5E2, BioLegend), CD11c (B-ly6, BD Pharmingen), CD303 (AC144, Miltenyi Biotec), and CD163 (5C6-FAT, Acris Antibodies).

Ohmatsu H., Humme D., Gulati N., Gonzalez J., Möbs M., Suárez-Fariñas M., Cardinale I., Mitsui H., Guttman-Yassky E., Sterry W, & Krueger J.G. (2014). Interleukin-32 is progressively expressed in Mycosis Fungoides independent of helper T-cell 2 and helper T-cell 9 polarization. Cancer immunology research, 2(9), 890-900.

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Immunophenotyping of Classic AT Patients

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Peripheral blood samples and clinical data were collected from 5 classic AT patients, who are under follow up at the Radboud University Medical Center (Table S1). Ethical approval was obtained from the Ethical Committee Arnhem/Nijmegen with protocol registration number 2011/304. Written informed consent was obtained from all patients. Five healthy control subjects were also included in our study (B17.001). Leukocyte subsets (absolute leukocyte counts and differential) were measured by using a Sysmex hematology analyzer and Trucount tubes (Becton Dickinson (BD), Franklin Lakes, NJ, USA) with a mixture of antibodies specific for lymphocyte subsets in freshly collected blood samples. Antibodies included CD45 (2D1, BD), CD3 (SK7; BD), CD19 (J4-119, Beckman Coulter, Brea, CA, USA), CD16 (3G8, BD) and CD56(C5.9, DAKO, Glostrup, Denmark). Samples were measured on a BD CANTO flow cytometer and analyzed with BD DIVA software. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation and cryo-stored before use.

Weitering T.J., Melsen J.E., van Ostaijen-ten Dam M.M., Weemaes C.M., Schilham M.W, & van der Burg M. (2021). Normal Numbers of Stem Cell Memory T Cells Despite Strongly Reduced Naive T Cells Support Intact Memory T Cell Compartment in Ataxia Telangiectasia. Frontiers in Immunology, 12, 686333.

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FACS Surface Staining of Immune Cells

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For FACS surface staining, the cells were labelled by the following anti-human antibodies: CD3 (SK7; BD), CD19 (SJ25C1; BD), CD123 (FAB301C; R&D Systems), CD11b (DCIS1/18), CD11c (BU15; Abcam), CD4 (A161A1, BD), CD8 (RPA-T8), FcεRI (AER-37 ([CRA-1]), CD14 (MϕP9; BD), CD45 (H130), CD56 (B159), CD45 (HI30, BD), CRTH2 (BM16; Miltenyi Biotec), IL-7Rα (A019D5), and live/dead violet (Invitrogen). The samples were acquired using FACSDiva on LSRFortessa flow cytometer. FlowJo software (FlowJo_v10.7.1) was used for data analysis.

Fonseka C.L., Hardman C.S., Woo J., Singh R., Nahler J., Yang J., Chen Y.L., Kamaladasa A., Silva T., Salimi M., Gray N., Dong T., Malavige G.N, & Ogg G.S. (2022). Dengue virus co-opts innate type 2 pathways to escape early control of viral replication. Communications Biology, 5, 735.

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Comprehensive Immune Profiling Post-Transplant

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Immune reconstitution was evaluated for several individuals 10-12 weeks after transplantation and peripheral blood, spleen, bone marrow, and thymus were analyzed using the following mouse mAbs that were specific for human epitopes and not cross-reactive with murine protein: CD62L(Dreg56)-FITC, CD25(2A3)-APC, CD3(SK7)-PerCP, CD8(SK1)-PerCP, CD8(SK1)-APC-H7, CD4(SK3)-FITC, CD4(SK3)-PerCP, CD14(M5E2)-PE, CD56(My31)-PE, HLA-DR(L243)-PerCP, NKp30(P30-15)-PE, NKp44(P44-8)-APC, NKp46(9E2)-PE and their corresponding IgG isotypes (BD PharMingen, Germany); CD45(MEM-28)-Pacific Blue, CD19(HIB19)-PerCP, CD3(MEM-57)-Alexa Fluor 700, and CD4(MEM-241)-Alexa Fluor 700 (Exbio, Czechoslovakia); CD45(HI30)-PE with respective isotype IgG (Biolegend, Germany). Engraftment was routinely checked 12-14 weeks after transplantation by retro-orbital bleeding and FACS staining. The A204 cell line was characterized with antibodies against ULBP-1(Z-9), ULBP-2(2F9), ULBP-3(F16), ULBP-4(6E6) (Santa Cruz Biotechnology, USA); MICA/B(6D4)APC CD112(R2.525)PE, CD155(SKII.4)PE (Biolegend, Germany); HLA-ABC(W6/32)PE (DAKO Cytomation, Germany); secondary antibody RAM-PE(X56) (BD PharMingen, Germany); and isotype controls (Beckman Coulter and R&D Systems, Germany). Flow cytometry was performed on an LSR II (BD Biosciences) using Diva© software.

Schilbach K., Alkhaled M., Welker C., Eckert F., Blank G., Ziegler H., Sterk M., Müller F., Sonntag K., Wieder T., Braumüller H., Schmitt J., Eyrich M., Schleicher S., Seitz C., Erbacher A., Pichler B.J., Müller H., Tighe R., Lim A., Gillies S.D., Strittmatter W., Röcken M, & Handgretinger R. (2015). Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation. Oncoimmunology, 4(7), e1014760.

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Activation and Phenotyping of T Cells

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Patients’ and healthy control (HC) PBMCs were purified by Ficoll density gradient centrifugation, washed with RPMI 1640 plus penicillin, streptomycin, and L-glutamine along with 10% FBS (R10) and filtered through a 40 µM strainer. Cells were resuspended to a concentration of 1 × 10⁶ cells/mL and aliquoted (1 mL/well) into 48-well plates. PMA (final concentration 20 ng/mL), ionomycin (1 µM) and brefeldin A (5 µg/mL, Sigma-Aldrich) were added prior to incubation at 37°C for 5–6 hours. After incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue (Life Technologies). Intracellular staining was performed using BD CytoFix/CytoPerm (BD Biosciences) reagents according to the manufacturer’s instructions using the following antibodies: CD3 (SK7, BD Biosciences), CD4 (RPA-T4, BD Biosciences), CD8 (SK1, BD Biosciences), CD45RO (UCHL1, Beckman Coulter), CXCR5 (RF8B2, BD Biosciences), CD25 (M-A251, BD Biosciences), IL-2 (MQ1–17H12, BioLegend), IL-4 (8D4–8, BD Biosciences), IL-5 (JES1–39D10, BD Biosciences), IL-13 (JES10-5A2, BD Biosciences), IFNγ (B27, BD Biosciences), IL-17A (eBio64DEC17, eBioscience). Cells were gated on viable CD3⁺ CD4⁺ CD45RO⁺ cells. Ex-vivo Treg staining was performed using anti-CD3 (UCHT1), CD4 (OKT4), CD25 (M-A251),, CD45RO (UCHL1), CD127 (HIL-7R–M21),, GATA3 (L50–823) and FOXP3 (236A/E7) antibodies as described ^{33 (link)}.

Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.

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CARMIL2-expressing T cell analysis

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We identified the T cell populations in which CARMIL2 reexpression occurred by staining PBMCs with the Abs against the following: CD3 (SK7), CD4 (SK3), CD8 (RPA-T8), CD25 (M-A251, all from BD), CD27 (O323; eBioscience), CD45RA (HI100, BD), CD56 (HCD56; Biolegend), FoxP3 (PCH101; eBioscience), and CARMIL2 (EM53; Exbio). Viability was assessed with Zombie Aqua Live/Dead stain (Biolegend). In patients with CARMIL2-expressing T cell populations, T lymphoblasts were generated by stimulating 10⁶ PBMCs with 5 ng/ml PMA, 1 µM ionomycin, and 100 U/ml IL-2 for 2 d before further expansion for 8–16 d in complete RPMI 1640 supplemented with 100 U/ml IL-2. The T lymphoblasts were sorted with Abs against CD3 (SK7), CD4 (SK3), CD8 (RPA-T8, all from BD), and CARMIL2 (EM53; Exbio). DNA was extracted from the CARMIL2-expressing cell populations with the DNeasy Blood and Tissue kit (Qiagen), and reversion events were confirmed by Sanger sequencing.

Lévy R., Gothe F., Momenilandi M., Magg T., Materna M., Peters P., Raedler J., Philippot Q., Rack-Hoch A.L., Langlais D., Bourgey M., Lanz A.L., Ogishi M., Rosain J., Martin E., Latour S., Vladikine N., Distefano M., Khan T., Rapaport F., Schulz M.S., Holzer U., Fasth A., Sogkas G., Speckmann C., Troilo A., Bigley V., Roppelt A., Dinur-Schejter Y., Toker O., Bronken Martinsen K.H., Sherkat R., Somekh I., Somech R., Shouval D.S., Kühl J.S., Ip W., McDermott E.M., Cliffe L., Ozen A., Baris S., Rangarajan H.G., Jouanguy E., Puel A., Bustamante J., Alyanakian M.A., Fusaro M., Wang Y., Kong X.F., Cobat A., Boutboul D., Castelle M., Aguilar C., Hermine O., Cheminant M., Suarez F., Yildiran A., Bousfiha A., Al-Mousa H., Alsohime F., Cagdas D., Abraham R.S., Knutsen A.P., Fevang B., Bhattad S., Kiykim A., Erman B., Arikoglu T., Unal E., Kumar A., Geier C.B., Baumann U., Neven B., Rohlfs M., Walz C., Abel L., Malissen B., Marr N., Klein C., Casanova J.L., Hauck F, & Béziat V. (2022). Human CARMIL2 deficiency underlies a broader immunological and clinical phenotype than CD28 deficiency. The Journal of Experimental Medicine, 220(2), e20220275.

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Comprehensive Immunophenotyping of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll step gradient and stained for cell surface markers with the following fluorochrome-conjugated antibodies: CD38 (HIT-2, BD), IgD (IA6-2, BD), IgM (MHM-88, BioLegend), CD10 (HI10a, BD), CD19 (SJ25C1, BD), CD21 (B-ly4, BD), CD27 (0323, eBiosience), CD138 (B-B4, AbD SeroTec), IgG (SouthernBioTech #2043-09), IgA (G20-359, BD), CD3 (SK7, BD), CD28 (CD28.2, BD), FOXP3 (PCH101, eBioscience), CD8 (SK1, BD), CD4 (RPA-T4, eBioscience), γδ-TCR (B1.1, eBioscience), αβ-TCR (IP26, eBioscience), CD45RO (UCHL1, BD), CD45RA (HI100, BD), CD57 (NK-1, BD), CD31 (WM59, eBioscience), CD62L (DREG-56, BD), CD152/CTLA4 (BNI3, BD). Samples were acquired on a FACS-Canto II flow cytometer (BD) or on a Gallios flow cytometer (Beckman Coulter, Miami, FL) and analyzed using FlowJo version 7.6.5 analysis software (Treestar, Ashland, OR).

Schubert D., Bode C., Kenefeck R., Hou T.Z., Wing J.B., Kennedy A., Bulashevska A., Petersen B.S., Schäffer A.A., Grüning B.A., Unger S., Frede N., Baumann U., Witte T., Schmidt R.E., Dueckers G., Niehues T., Seneviratne S., Kanariou M., Speckmann C., Ehl S., Rensing-Ehl A., Warnatz K., Rakhmanov M., Thimme R., Hasselblatt P., Emmerich F., Cathomen T., Backofen R., Fisch P., Seidl M., May A., Schmitt-Graeff A., Ikemizu S., Salzer U., Franke A., Sakaguchi S., Walker L.S., Sansom D.M, & Grimbacher B. (2014). Autosomal-dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nature medicine, 20(12), 1410-1416.

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FACS Staining of Human Immune Cells

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For FACS surface staining the cells were labelled by the following anti human antibodies purchased from Biolegend unless stated otherwise: CD3 (SK7; BD Biosciences), CD19 (SJ25C1; BD Biosciences), CD123 (FAB301C; R&D systems), CD11b (DCIS1/18), CD11c (BU15; Abcam), CD8 (RPA-T8), FcεRI (AER-37 (CRA-1)), CD14 (MφP9; BD biosciences), CD4 (MEM-241), CD45 (H130), ICOS (C398.4A), CD56 (B159), CRTH2 (BM16; Miltenyibiotec), IL-7Rα (A019D5), live/dead violet (L34955; Invitrogen), NKp30 (clone: AF29-4D12), NKp30 blocking antibody (Clone 210845 R&D systems, AF29-4D12 Miltenyi Biotec), Phospho-IκBα (Ser32/36 Cell Signalling 9246), Anti-B7-H6 antibody (ab121794), B7-H6 blocking antibody (17BL.3), CD68 (Y1/82A), Siglec-8 (7C9) and CD16 (3G8).

Salimi M., Xue L., Jolin H., Hardman C., Cousins D.J., McKenzie A.N, & Ogg G.S. (2015). Group 2 innate lymphoid cells express functional NKp30 receptor inducing type 2 cytokine production. Journal of immunology (Baltimore, Md. : 1950), 196(1), 45-54.

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Phenotyping Antigen-Specific B cells

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Freshly isolated peripheral blood mononuclear cells were stained first for viability with Live/dead Yellow (ThermoFisher) and then for markers with the following monoclonal antibodies: IgA (IS11–8E10, Miltenyi), IgD (IA6–2, BD), IgG (G18–145, BD), IgM (MHM-88, Biolegend), CD3 (SK7, BD), CD4 (RPA-T4, BD), CD8 (SK1, BD), CD14 (61D3, eBioscience), CD16 (CB16, eBioscience), CD19 (SJ25C1, BD), CD20 (2H7, BD), CD27 (O323, BioLegend or M-T271, BD), CD38 (HB7, BD), and CD71 (CY1G4, BioLegend. Antigen-specific B cells were detected by staining with RBD conjugated to Alexa Fluor 488 (Protein Labeling Kit, ThermoFisher). RBD was conjugated according to manufacturer’s instructions, with the following changes: protein was labeled at a concentration of 1mg/mL, and incubated for 30 minutes without the addition of bicarbonate. After staining, PBMCs were washed and then fixed for 30 minutes using 2% paraformaldehyde (ThermoFisher). Data were acquired on a BD FACSymphony A5 and analyzed using FlowJo 10.7.1 (BD).

Mantus G., Nyhoff L.E., Kauffman R.C., Edara V.V., Lai L., Floyd K., Shi P.Y., Menachery V.D., Edupuganti S., Scherer E.M., Kay A., McNair N., Anderson E.J., Rouphael N., Ahmed R., Suthar M.S, & Wrammert J. (2021). Evaluation of Cellular and Serological Responses to Acute SARS-CoV-2 Infection Demonstrate the Functional Importance of the Receptor Binding Domain. Journal of immunology (Baltimore, Md. : 1950), 206(11), 2605-2613.

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