Anti cd8a clone 53 6

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD8a (clone 53-6.7) is a monoclonal antibody that binds to the CD8a molecule, which is expressed on the surface of certain T cells. This antibody can be used for the identification and enumeration of CD8+ T cells in flow cytometry applications.

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FITC-anti-mouse CD8a (clone 53-6.7, BV605-anti-CD8a (clone 53–6.7). FITC-conjugated anti-mouse CD8a (clone 53–6.7, CD8a (53-6.7, Clone 53-6.7 Anti-CD8a

5 protocols using anti cd8a clone 53 6

Immune Activation Experiment Protocols

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For immune activation experiments in vivo, ascitic cells were harvested and washed twice with PBS and incubated with the following antibodies: anti-NK1.1 (clone PK136, #553164), CD11b (clone M1/70, #557396), Gr-1 (clone RB6-8C5, #553128) (all from BD Biosciences, Franklin Lakes, NJ, USA); and anti-CD3e (clone 17A2, #17-00032-82), anti-CD8a (clone 53-6.7, #45-0081-82), and isotype antibodies (#45-4321-80, 11-4714-41, 17-4031-81, and 12-4321-81A) (all from Thermo Fisher Scientific, eBioscience). CD11b⁺Gr-1^int cells were defined as MDSCs, and CD3⁺CD8⁺ cells were defined as CD8⁺ T cells (cytotoxic T lymphocytes). Samples were subjected to flow cytometry using a FACS Calibur instrument (BD Biosciences), and data were analyzed using FlowJo software (v. 7.6.5, Tree Star, Ashland, OR, USA).

Meng G., Fei Z., Fang M., Li B., Chen A., Xu C., Xia M., Yu D, & Wei J. (2019). Fludarabine as an Adjuvant Improves Newcastle Disease Virus-Mediated Antitumor Immunity in Hepatocellular Carcinoma. Molecular Therapy Oncolytics, 13, 22-34.

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Multiparametric Flow Cytometry of Splenocytes

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Surface antigens on splenocytes were stained using anti-CD169 (clone MOMA-1), anti-CD11c (clone N418), anti-CD8a (clone 53-6.7, eBioscience), anti-CD4 (clone L3T4, eBioscience), anti-F4/80 (clone BM8, eBioscience), and fixable viability dye (eBioscience). Cells were analyzed using the flow cytometer LSR Fortessa (Becton Dickinson).

Friedrich S.K., Schmitz R., Bergerhausen M., Lang J., Duhan V., Hardt C., Tenbusch M., Prinz M., Asano K., Bhat H., Hamdan T.A., Lang P.A, & Lang K.S. (2021). Replication of Influenza A Virus in Secondary Lymphatic Tissue Contributes to Innate Immune Activation. Pathogens, 10(5), 622.

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Isolation and Characterization of Murine Hematopoietic Stem Cells

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Mononuclear BM cells were isolated by low-density centrifugation (Histopaque 1083, Sigma) and stained with a cocktail of biotinylated lineage antibodies. Biotinylated antibodies used for lineage staining were all rat anti-mouse antibodies: anti-CD11b (clone M1/70), anti-B220 (clone RA3-6B2), anti-CD5 (clone 53-7.3), anti-Gr-1 (clone RB6-8C5), anti-Ter119 and anti-CD8a (Clone 53-6.7) (all from eBioscience). After lineage depletion by magnetic separation (Dynalbeads, Invitrogen), cells were stained with anti-Sca-1 (clone D7) (eBioscience), anti-c-kit (clone 2B8) (eBioscience), anti-CD34 (clone RAM34) (eBioscience), anti-CD127 (clone A7R34) (eBioscience), anti-Flk-2 (clone A2F10) (eBioscience), and Streptavidin (eBioscience). Early haematopoiesis FACS analysis data were plotted as percentage of long-term haematopoietic stem cells (LT-HSCs, gated as LSK CD34^−/lowFlk2⁻), short-term haematopoietic stem cells (ST-HSCs, gated as LSK CD34⁺Flk2⁻), and lymphoid-primed multipotent progenitors (LMPPs, gated as LSK CD34⁺Flk2⁺) distribution among LSKs (Lin^negc-kit⁺sca-1⁺ cells). In order to isolate HSCs, lineage depletion was performed to enrich for lineage negative cells. Lineage negative cells were then stained as aforementioned and sorted using a BD FACS Aria III (BD Bioscience). More information on the antibodies used is provided in the Source Data file.

Montserrat-Vazquez S., Ali N.J., Matteini F., Lozano J., Zhaowei T., Mejia-Ramirez E., Marka G., Vollmer A., Soller K., Sacma M., Sakk V., Mularoni L., Mallm J.P., Plass M., Zheng Y., Geiger H, & Florian M.C. (2022). Transplanting rejuvenated blood stem cells extends lifespan of aged immunocompromised mice. NPJ Regenerative Medicine, 7, 78.

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Multicolor Flow Cytometry Analysis

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The following monoclonal antibodies were used: FcR‐blocking antibody (anti‐mouse CD16/CD32, clone 2.4G2; BD Pharmingen), FITC, PE, PerCP, or PerCP/Cy5.5‐conjugated anti‐CD335 (clone 29A1.4; BioLegend), anti‐CD49b (clone HMα2; BioLegend), anti‐CD11b (clone M1/70; BioLegend), anti‐CD4 (clone RM4‐5; eBioscience), anti‐CD8a (clone 53‐6.7; eBioscience), anti‐CD45.2 (clone 104; BioLegend), anti‐IFN‐γ (clone XMG1.2; BioLegend), Rat IgG2a,k (BD Pharmingen), Rat IgG2b, k control (BD Pharmingen), Rat IgG1k isotype‐matched control Abs (BioLegend), and PE/Cy7‐conjugated Armenian hamster IgG (BioLegend) Abs.
Cells were labeled at 4°C with specific antibodies. For intracellular cytokine analysis, cells were resuspended in RPMI‐1640 medium supplemented with 20% FBS, and phorbol 12‐myristate 13‐acetate (50 ng/mL), ionomycin (500 ng/mL), and brefeldin A (10 μg/mL; all from Sigma) were added. After incubation for 4 h, the cells were washed, stained for surface molecule markers, and fixed and permeabilized using IntraPrep reagent (Beckman Coulter), followed by intracellular staining of IFN‐γ. The stained cells were analyzed using a FACSCalibur (BD Biosciences).

Umemoto S., Haruta M., Sakisaka M., Ikeda T., Tsukamoto H., Komohara Y., Takeya M., Nishimura Y, & Senju S. (2019). Cancer therapy with major histocompatibility complex‐deficient and interferon β‐producing myeloid cells derived from allogeneic embryonic stem cells. Cancer Science, 110(10), 3027-3037.

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Isolation and Sorting of Murine and Human Hematopoietic Stem Cells

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Low‐density mononuclear cells were isolated by density centrifugation (Histopaque 1083, Sigma) and stained with a cocktail of biotinylated lineage antibodies all rat anti‐mouse antibodies: anti‐CD11b (clone M1/70), anti‐B220 (clone RA3‐6B2), anti‐CD5 (clone 53‐7.3) anti‐Gr‐1 (clone RB6‐8C5), anti‐Ter119 and anti‐CD8a (clone 53‐6.7; eBioscience). After lineage depletion by magnetic separation (Dynabeads, Invitrogen), cells were stained with anti‐Sca‐1 (clone D7), anti‐c‐Kit (clone 2B8), anti‐CD34 (clone RAM34), anti‐Flk2 (clone A2F10), and Streptavidin antibodies (eBioscience). Stained HSCs (gated as Lin⁻Sca⁺Kit⁺CD34⁻Flk2⁻) were sorted in 96 well plates using a BD FACS Aria II (BD Bioscience) as previously reported (Florian et al, 2012 (link)). Human BM MNCs were enriched using standard Ficoll‐based density centrifugation in the clinical laboratory at CCHMC. Human HSPCs (lin^neg CD34^pos SSC^low cells from BM) were FACS‐sorted (BD Aria II, BD Bioscience) according to standard protocols (sorted lin^neg CD34^pos SSC^low cells from BM, anti‐CD34 clone 581 (BD #555824)), human hematopoietic lineage antibody cocktail, FITC, eBioscience (#22‐7778‐72; Kumar et al, 2021 (link)).

Kumar S., Vassallo J.D., Nattamai K.J., Hassan A., Karns R., Vollmer A., Soller K., Sakk V., Sacma M., Nemkov T., D'Alessandro A, & Geiger H. (2023). pH regulates hematopoietic stem cell potential via polyamines. EMBO Reports, 24(5), e55373.

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