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Cd8 apc cy7

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The CD8-APC-Cy7 is a fluorescent-labeled antibody that binds to the CD8 surface antigen. It is designed for use in flow cytometry applications to identify and quantify CD8-positive cells within a sample.

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Lab products found in correlation

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Close spelling variants of this product

Anti-CD8-APC-Cy7 Anti-human CD8 APC-Cy7 (clone SK1) CD8-APC Cy7 (SK1, CD8 APC-Cy7 (53-6.7, APC-Cy7-conjugated anti-CD8 α-CD8-APC-Cy7 APC-Cy7-CD8 Ab APC-Cy7 anti-mouse CD8 APC-Cy7-labeled CD8 APC-cy7-labelled anti-CD8

90 protocols using cd8 apc cy7

1

Flow Cytometry Profiling of Lymphocyte Subsets

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Flow cytometry analysis was performed to quantify lymphocyte subsets on BD LSR II flow cytometer with BD FACS DIVA software (BD Biosciences, Heidelberg, Germany). The following antibodies were used for phenotypic analysis: CD45-FITC, CD19/20-PE, CD14-Cy-7, CD25-FITC, CD127-PE, CD45RA-APC, CD3-APC Cy-7, CD8-FITC, CD4-PE, CD4-APC Cy7, CD8-APC Cy7, CD62L-FITC, CCR7-PE, CD27-FITC, CD62L-PE, (Beckton Dickinson, Franklin Lakes, NJ, USA), CD3-ECD, CD56-APC, CD45RO-ECD (Beckman Coulter, Miami, FL, USA).30 (link)
Triplett B.M., Shook D.R., Eldridge P., Li Y., Kang G., Dallas M., Hartford C., Srinivasan A., Chan W.K., Suwannasaen D., Inaba H., Pui C.H, & Leung W. (2015). Rapid Memory T-cell Reconstitution Recapitulating CD45RA-depleted Haploidentical Transplant Graft Content in Patients with Hematologic Malignancies. Bone marrow transplantation, 50(7), 968-977.
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2

Sorting and Characterizing Naive and Memory T Cells

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Cryopreserved PBMC samples were labelled using CD4-PE, CD8-APC-Cy7, CD45RA-APC (all Becton Dickinson) and CD62L-ECD (Beckman Coulter) on ice, following overnight rest to restore CD62L expression (Figure A in S1 File). CD4+ and CD8+ gated lymphocytes were sorted (Influx; Becton Dickinson) on the basis of CD45RA and CD62L expression [20 (link)]. Sort purities were regularly >98%. We confirmed naïve and memory populations by stimulating naïve or memory CD8+ T cells with cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza derived peptides (CEF peptides; Mabtech) and performing an IFN-γ ELISpot assay as described above. CEF peptides responses were only detected in stimulated memory phenotype T cells, but not naïve populations (Figure B in S1 File).
Lucas A., Lucas M., Strhyn A., Keane N.M., McKinnon E., Pavlos R., Moran E.M., Meyer-Pannwitt V., Gaudieri S., D’Orsogna L., Kalams S., Ostrov D.A., Buus S., Peters B., Mallal S, & Phillips E. (2015). Abacavir-Reactive Memory T Cells Are Present in Drug Naïve Individuals. PLoS ONE, 10(2), e0117160.
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3

Flow Cytometry Profiling of Lymphocyte Subsets

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Flow cytometry analysis was performed to quantify lymphocyte subsets on BD LSR II flow cytometer with BD FACS DIVA software (BD Biosciences, Heidelberg, Germany). The following antibodies were used for phenotypic analysis: CD45-FITC, CD19/20-PE, CD14-Cy-7, CD25-FITC, CD127-PE, CD45RA-APC, CD3-APC Cy-7, CD8-FITC, CD4-PE, CD4-APC Cy7, CD8-APC Cy7, CD62L-FITC, CCR7-PE, CD27-FITC, CD62L-PE, (Beckton Dickinson, Franklin Lakes, NJ, USA), CD3-ECD, CD56-APC, CD45RO-ECD (Beckman Coulter, Miami, FL, USA).30 (link)
Triplett B.M., Shook D.R., Eldridge P., Li Y., Kang G., Dallas M., Hartford C., Srinivasan A., Chan W.K., Suwannasaen D., Inaba H., Pui C.H, & Leung W. (2015). Rapid Memory T-cell Reconstitution Recapitulating CD45RA-depleted Haploidentical Transplant Graft Content in Patients with Hematologic Malignancies. Bone marrow transplantation, 50(7), 968-977.
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4

Peptide-specific T cell analysis

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Peptides p53(264-272), gp100(280-288), MDM2(81-88), and pp65(495-503) were synthesized by Biosyntan (Berlin) or PSL (Heidelberg, GER). The monoclonal antibodies (mAbs) used were against Hu CD4-, or CD8-FITC, Hu Pan TCRαβ-, Hu Vβ13.1-, Vβ14-PE (Beckman-Coulter, Krefeld), Hu CD3(cl. HIT3a)-FITC, CD8-APC-Cy7, Mu TCRβ-, Vβ3-PE, Hu IFNγ-APC (BD Biosciences, Heidelberg, GER). PE- or PE-Cy7-labeled tetrameric p53-specific or gp100 pA2.1 complexes were obtained from LICR (Epalinges, Switzerland), pp65(495-503) tetramers were from Beckman-Coulter, gp100(280-288)-specific dextramers from Immudex (Copenhagen, Denmark).
Knies D., Klobuch S., Xue S.A., Birtel M., Echchannaoui H., Yildiz O., Omokoko T., Guillaume P., Romero P., Stauss H., Sahin U., Herr W., Theobald M., Thomas S, & Voss R.H. (2016). An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells. Oncotarget, 7(16), 21199-21221.
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5

Flow Cytometric Analysis of Splenic T Cells and Anti-MUC1 Antibodies

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Spleens were collected at necropsy and a single cell suspension was obtained by passing the tissue fragments through a 70 µm cell strainer (BD Falcon, Franklin Lake, NJ, USA). Cells were stained with fluorescent antibodies for CD3 (PerCP), CD4 (Pacific Blue), and CD8 (APC-Cy7) (all antibodies from BD Biosciences, San Jose, CA), followed by intracellular staining for Foxp3 (eBioscience, San Diego, CA), according to the manufacturers' protocols.
To detect anti-MUC1 antibodies, samples were incubated with IG10-MUC1 cells [29] (link) expressing extracellular human MUC1. To detect bound antibodies, the cells were then stained with fluorescein tagged anti-mouse IgG and positive cells analyzed with LSRII (BD Biosciences) and processed in FACSDiva (BD Biosciences). Gates for positive cells were set using control ascites, from tumor bearing KrasPten (i.e. human MUC1 negative) mice.
Tirodkar T.S., Budiu R.A., Elishaev E., Zhang L., Mony J.T., Brozick J., Edwards R.P, & Vlad A.M. (2014). MUC1 Positive, Kras and Pten Driven Mouse Gynecologic Tumors Replicate Human Tumors and Vary in Survival and Nuclear Grade Based on Anatomical Location. PLoS ONE, 9(7), e102409.
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6

Multiparametric Immune Profiling of Cells

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Aliquots of 1 × 106 isolated liver-derived cells and PBMC were stained for surface markers with pre-diluted fluorochrome-conjugated anti-human monoclonal antibodies: BD Multitest™ CD16-PE (IgG1, clone B73.1) and CD56-PE (IgG1, clone NCAM 16.2); CD3/CD16+CD56/CD45/CD19 reagent (CD3-FITC (IgG2a, clone SK7); CD45-PerCP (IgG1, clone 2D1); CD335/NKp46-PE-Cy7 (IgG1, clone 9E2/Nkp46); CD19-APC (IgG1, clone SJ25C1)) and CD3-FITC (IgG2a, clone SK7); CD4-PE-Cy7 (IgG1, clone L200); CD56-AlexaFluor-700 (IgG1, clone B159); CD8-APC-Cy7 (IgG1, clone SK1); antiCD337/NKp30-AlexaFluor-647 (IgG1, clone p30-15); and CD314/NKG2D-PerCP-Cy5.5 (IgG1, clone 1D11). These antibodies were purchased from BD Biosciences (Europe, dilution 1:10). CD161-FITC (IgG2a, clone 191B8); CD3-PerCP (IgG2a, clone BW264/56); CD16-APC (IgM, clone VEP13); and CD336/NKp44-PE (IgG1, clone 2.29) from Miltenyi Biotec (Bergisch Gladbach, Germany, dilution 1:20). TCRVa7.2-PE (IgG1, clone 3C10) was obtained from Biolegend (Campoverde s.r.l., Milan, Italy, dilution 1:25). At room temperature in the dark, LP samples were incubated for 30 min, and washed once with PBS/2% FBS. They were analyzed by flow cytometry with a FACSAria II cytometer (BD Biosciences, Eysins, Switzerland).
Pagano D., Badami E., Zito G., Conaldi P.G., Vella I., Buscemi B., Amico G., Busà R., Salis P., Li Petri S., di Francesco F., Calamia S., Bonsignore P., Tropea A., Accardo C., Piazza S, & Gruttadauria S. (2023). Impact of T Lymphocytes Isolated from Liver Perfusate of Deceased Brain Donors on Kidney Transplantation: Preliminary Evidence and Future Directions. Journal of Clinical Medicine, 12(14), 4786.
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7

Multiplex cytokine analysis of Her2-specific T-cells

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PBMCs were thawed, washed, counted and re-suspended in X-Vivo 15 medium supplemented with IL-4 (5 ng/ml) and IL-7 (5 ng/ml) on day 0. On d1, pooled 15-mer Her-2 peptides (PepMix, JPT Technologies, Berlin) were added at 1 µg/ml to 1 × 106 cells per culture. IL-2 (40U/ml) was added on d3. T-cells were harvested on d12 and re-stimulated (0.4–0.5 × 106 cells/well) with 1 µg/ml Her2 peptides or left un-stimulated as a negative control for 12 h. Pepmixes of influenza nucleoprotein and matrix protein were used as positive controls. Golgi-plug (BD-Biosciences) was added at 1 µl/ml to prevent cytokine secretion. The cells were harvested, washed, incubated with Gamunex and EMA, fixed and permeabilized with Cytofix/Cytoperm (BD-Biosciences) before staining with CD3-Pacific Orange (Invitrogen), CD4-Pacific Blue, TNF-FITC, IL-2-Alexa-Fluor-700, IL-5-PE (BioLegend), CD8-APC-Cy7, IFN-γ-PE-Cy7 (BD-Biosciences), IL-10-APC (Miltenyi-Biotec) and IL-17-PerCP-Cy5.5 (eBioscience). After washing, the cells were immediately measured using a BD-LSR-II flow cytometer with FACS-Diva software (BD-Biosciences).
Kini Bailur J., Gueckel B, & Pawelec G. (2016). Prognostic impact of high levels of circulating plasmacytoid dendritic cells in breast cancer. Journal of Translational Medicine, 14, 151.
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8

Profiling Colorectal Immune Cells

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7 weeks before the first viral challenge, an open laparotomy was performed on each of the vaccinated and adjuvanted animals to obtain a colorectal wedge. For the naive animals, 10 rectal pinches were collected. The colonic wedge was rinsed with HBSS to remove the mucus, and then cut into 2″ pieces. The colonic tissue pieces/rectal pinches were incubated with HBSS containing 5 mM EDTA and 2 mM DTT at room temperature for 15 min twice. The supernatants, which contain intraepithelial lymphocytes (IEL) cells, were collected, pooled and filtered through 100 μM cell strainers. The collected colorectal IELs were washed and subjected to FACS staining with viability dye and antibody mixtures including anti-CD45 to exclude the dead and CD45 negative epithelial cells. The frequencies of ki67, CD69, CD38 and HLA-DR within CD4+ and CD8+ T cells were further assessed. About 8000, 6000, and 2000 CD4+T cells (mean) were analyzed for vaccinated, adjuvanted and naive animals (Supplementary Fig. 1). The following antibodies were purchased: CD3-PE-Cy7, CD8-APC-Cy7, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (from BD Pharmingen); CD69-Alexa Fluor 700 (from Biolegend); CD38-FITC (from STEMCELL Technologies); and CD4-qdot 605 (from eBioscience).
Sui Y., Lee E.M., Wang Y., Hogg A., Frey B., Venzon D., Pal R, & Berzofsky J.A. (2016). Early SIV dissemination after intra-rectal SIVmac251 challenge was associated with proliferating virus-susceptible cells in the colorectum. Journal of acquired immune deficiency syndromes (1999), 71(4), 353-358.
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9

Detailed Flow Cytometry Immunophenotyping

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Mouse thymocytes and splenocytes were isolated in RPMI containing 5% fetal calf serum, followed by red blood cell depletion using the ACK lysing buffer (Gibco). Flow cytometric analysis was performed on cells according to a previously described procedure (Jiang et al., 2011 (link)). Antibodies used for analyses: CD3 pacific blue (BD, clone 500A2, 558214), CD8 APC-Cy7 (BD, clone 53-6.7, Cat. No. 557654), CD4 PE (BD, clone GK1.5, Cat. No. 553730), CD80 FITC (BD, Cat. No. 553768), mouse Vβ TCR screen panel (BD, Cat. No. No. 557004), CD25 APC (eBioscience, clone PC61.5, Cat. No. 17-0251-82), Foxp3 FITC (eBioscience, clone FJK16a, Cat. No. 11-5773-82), CD3e FITC (eBioscience, clone 145-2C11, 11-0031-82), CD274 PE (eBioscience, B7-H1, PD-L1, clone MIH5, 12-5982-81), ly-6G (Gr-1) Alexa Fluor 700 (eBioscience, clone RB6-8c5, 56-5931-80), CD11b PE-Cy7 (eBioscience, clone M1/70, 25-0112-81), CD11c 780 (eBioscience, clone N418, 47-0114-80), CD49f (intergrin alpha 6) PE-Cyanine 7 (eBioscience, clone GOH3, Cat. No. 25-0495-80), CD45.1 PE-Cy7 (eBioscience, clone A20, 25-0453-82), CD45.2 eFluor 450 (eBioscience, clone 104, Cat# 48-0454-82), and FITC conjugated UEA1 (Vector, Cat. No. FL-1061). Labeled cells were analyzed on a LSR-II Flow Cytometer (Becton Dickinson). Data were analyzed using FlowJo software.
Zhu F., Willette-Brown J., Song N.Y., Lomada D., Song Y., Xue L., Gray Z., Zhao Z., Davis S.R., Sun Z., Zhang P., Wu X., Zhan Q., Richie E.R, & Hu Y. (2017). Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis. Cell host & microbe, 21(4), 478-493.e7.
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10

EBOV GP-Specific T Cell Responses

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ICS assays were performed as previously described5 (link). In brief, freshly isolated mouse splenocytes or monkey PBMCs were seeded into 96-well plates (2 × 106 cells per well) and incubated with an EBOV GP peptide pool (2 µg/ml). One hour later, brefeldin A (BD Phamingen, CA, USA) was added and incubated for another 10 h. The cells were then harvested, stained with surface antibodies (CD3-Pacific blue, CD8-APC-Cy7, CD4-FITC; BD Biosciences, CA, USA) for 1 h, then permeabilized and stained with intracellular antibodies (IFN-γ-PE, IL-2-APC, TNF-α-PE-Cy7; BD Biosciences, CA, USA). Finally, the cells were detected with an LSR Fortessa SORP instrument (BD Biosciences, CA, USA).
Feng Y., Li C., Hu P., Wang Q., Zheng X., Zhao Y., Shi Y., Yang S., Yi C., Feng Y., Wu C., Qu L., Xu W., Li Y., Sun C., Gao F.G., Xia X., Feng L, & Chen L. (2018). An adenovirus serotype 2-vectored ebolavirus vaccine generates robust antibody and cell-mediated immune responses in mice and rhesus macaques. Emerging Microbes & Infections, 7, 101.
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