Cd8a apc h7

Manufactured by BD

Sourced in Austria

CD8a-APC-H7 is a fluorochrome-conjugated antibody that binds to the CD8a cell surface marker. It is used for the identification and enumeration of CD8+ T cells in flow cytometry applications.

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CD8a-APC (10 citations) APC-H7 rat anti-mouse CD8a (2 citations)

4 protocols using cd8a apc h7

Tumor-Infiltrating Immune Cell Analysis

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M-chlorin e6 PDT was performed as described above. Mice were euthanized by cervical dislocation on day 2 after LED irradiation. The tumors were removed and treated with 10% collagenase (Wako), shredded using a gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and passed through a 70-μm filter. Next, 1.0 × 10⁶ cells were mixed with 1 μL of Fc-block/100 μL of stain buffer (BD Biosciences, San Jose, CA) and left on ice for 10 min. Subsequently, 1 μL of fluorescent antibody (CD11b-APC [BD Biosciences], CD206-BV421 [Biolegend, San Diego, CA], CD80-PE [BD Biosciences], CD86-PE-Cy7 [BD Biosciences], CD45-PE-Vio®770 [Miltenyi Biotec], CD3-FITC [Miltenyi Biotec], CD8a-APC-H7 [BD PharMingen, San Diego, CA], CD127-APC [BD PharMingen], CD25-PE [BD Biosciences], or CD4-BV421 [Biolegend]) diluted with 100 μL of stain buffer was added and left on ice for 30 min. The cells were then washed with 500 μL of phosphate-buffered saline (PBS) and suspended in 500 μL of stain buffer. Next, 5 μL of 7-AAD (BD Biosciences) was added to prepare a sampling solution, which was analyzed on a BD FACSVerse™ (BD Biosciences).

Soyama T., Sakuragi A., Oishi D., Kimura Y., Aoki H., Nomoto A., Yano S., Nishie H., Kataoka H, & Aoyama M. (2021). Photodynamic therapy exploiting the anti-tumor activity of mannose-conjugated chlorin e6 reduced M2-like tumor-associated macrophages. Translational Oncology, 14(2), 101005.

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Splenocyte Immunophenotyping by Flow Cytometry

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After isolation and re-stimulation, the splenocytes were collected, washed and immunophenotyped by using the following anti-mouse mAbs: CD3-eFluor 450, CD4-FITC (Thermo Fisher Scientific), and CD8a-APC-H7 (BD Biosciences). Appropriate isotype controls were used for comparison. After staining, the splenocytes were assessed by BD FACSAria™ II Flow Cytometer (BD Biosciences, San Jose, CA, USA). Dead cells were excluded by using LIVE/DEAD Fixable Dead Cell Stain (Invitrogen). Antigen specific CD4⁺ or CD8⁺ T cells were analyzed with FlowJo software (Tree Star Inc., Ashland, OR) [43 (link)]. All experiments were performed in triplicate.

Bai X., Zhou Y., Yokota Y., Matsumoto Y., Zhai B., Maarouf N., Hayashi H., Carlson R., Zhang S., Sousa A., Sun B., Ghanbari H., Dong X, & Wands J.R. (2022). Adaptive antitumor immune response stimulated by bio-nanoparticle based vaccine and checkpoint blockade. Journal of Experimental & Clinical Cancer Research : CR, 41, 132.

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Isolation and Phenotyping of Tumor-Infiltrating Immune Cells

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Tumors were excised from the colon, cut into small pieces with a scalpel and washed with HBSS containing 20 mM HEPES and penicillin/streptomycin (P/S) 4 x 10 min at room temperature. Intraepithelial immunocytes were extracted by incubation with HBSS containing 10 mM EDTA, 2.5% FBS, and P/S for 4 x 20 min at 37°C on a rotating device. The tissue was washed with RPMI and digested with RPMI supplemented with 5% FBS, 10mM HEPES, P/S, and 200 U/ml collagenase type II for 1 hour at 37°C (all Life Technologies). Isolated cells were passed through a 40µm cell strainer, washed with PBS once, and leukocytes were stained for flow cytometry. Cells were stained with the following antibodies (all BD, Vienna, Austria) for 1 hour at 4°C: CD45-APC (1:200, clone: 30-F11), CD3e-BV510 (1:100, clone 145-2C11), CD4-PE-Cy7 (1:500, clone RM4-5), CD8a-APC-H7 (1:100, clone 53-6.7), CD11b-FITC (1:200, clone M1/70), Ly6G-BV421 (1:500, clone 1A8), Ly6C-PE-Cy7 (1:200, clone AL-21), CD16/32 (1:100, clone 2.4G2). Samples were analyzed on a BD FACS Canto II flow cytometer. Analysis was done with FlowJo 4.0 software.

Hasenoehrl C., Feuersinger D., Sturm E.M., Bärnthaler T., Heitzer E., Graf R., Grill M., Pichler M., Beck S., Butcher L., Thomas D., Ferreirós N., Schuligoi R., Schweiger C., Haybaeck J, & Schicho R. (2017). G protein-coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1. International journal of cancer, 142(1), 121-132.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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To examine different cell populations within human melanoma patient PBMC and mouse Pmel-1 splenocytes, cell surface flow cytometry was performed as previously described.27 (link) Human melanoma patient PBMC were stained with the following anti-human antibodies from BD: Fixable Viability Stain 575V, CD3 APC-R700, CD19 APC-H7, CD47 APC, PD-1 BV 421, and CD8 BUV496. Pmel-1 splenocytes were stained with dilutions of the following anti-mouse antibodies from BD: CD19 BV421, Fixable Viability Stain 575V, CD64 BV786, CD8a APC-H7, and CD3 BUV496. Flow cytometry was performed on the BD LSRFortessa X-20 Analyzer, and analysis was performed on FCS Express software (online supplemental figures 3C and 3D).

Stirling E.R., Terabe M., Wilson A.S., Kooshki M., Yamaleyeva L.M., Alexander-Miller M.A., Zhang W., Miller L.D., Triozzi P.L, & Soto-Pantoja D.R. (2022). Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response. Journal for Immunotherapy of Cancer, 10(11), e004712.

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