Cd8 antibody

Manufactured by BD

Sourced in United States

The CD8 antibody is a laboratory reagent used in flow cytometry to identify and quantify CD8-positive cells, such as cytotoxic T cells, within a sample. The antibody binds specifically to the CD8 surface marker, which is expressed on a subset of T lymphocytes. This allows for the detection and enumeration of these cells in various biological samples.

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7 protocols using cd8 antibody

Analyzing Mouse Immune Cell Phenotypes

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Mouse BMDCs or splenocytes were washed with PBS. For surface marker extracellular staining, the cells were incubated with anti-mouse CD11c (557,400, BD Biosciences), CD80 (560,526, BD Biosciences), CD86 (552,692, BD Biosciences), MHC-II (562,367, BD Biosciences), CD11b (101,211, Biolegend), CD4 (553,046, BD Biosciences), or CD8 antibodies (551,162, BD Biosciences) at 4 °C for 30 min. For intracellular cytokine staining, the splenocytes were stimulated with DnaJ (10 μg/ml) for 8 h in presence of Golgi plug™ (51-2301KZ, BD Bioscience). The splenocytes were then treated for surface markers (CD4 or CD8), fixed/permeabilised with a Cytofix/Cytoperm solution (51-2090KZ, BD Bioscience) and then stained with anti-IFN-γ (557,735, BD Biosciences) and anti-IL-4 (554,435, BD Biosciences) antibodies at 20–25 °C for 30 min. All events were acquired on a FACSverse flow cytometer and analysed using the FlowJoV software (Tree Star).
To determine cytokine (IFN-γ, TNF-α, IL-1β, IL-10, IL-17a, MCP-1, IL-1α, and IL-6) concentrations in the uterine tissue, multi-analyte flow assay kits (740,446, Biolegend, San Diego, CA, USA) were used as indicated by the manufacturer.

Guo F., Tang Y., Zhang W., Yuan H., Xiang J., Teng W., Lei A., Li R, & Dai G. (2022). DnaJ, a promising vaccine candidate against Ureaplasma urealyticum infection. Applied Microbiology and Biotechnology, 106(22), 7643-7659.

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Galectin-3 Inhibition Modulates CD8+ T Cell Activation

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The 24-well plates were coated with CD3 antibody (OKT3, Invitrogen, Carlsbad, CA, USA) overnight. PBMCs were isolated from the peripheral blood of healthy donors using Human PBMC Isolate kit (TBD, Tianjin, China). CD8⁺ T cells were positive selected from PBMC using magnetic beads coated with CD8 antibodies (BD, Franklin Lakes, NJ, USA). These CD8⁺ T cells were placed into the Transwell lower chamber along with CD28 antibody (Invitrogen, USA) and IL-2 (Procell, Wuhan, China). A549 or A549-3a cells were seeded in Transwell inserts (Corning, Corning, NY, USA). For certain experiments, cells were treated with the galactin-3 inhibitor (TD-139) (Selleck, Houston, TX, USA). The co-culture system was incubated for 48 h at 37 °C and 5% CO₂.

Wang Y., Yang C., Wang Z., Wang Y., Yan Q., Feng Y., Liu Y., Huang J, & Zhou J. (2023). Epithelial Galectin-3 Induced the Mitochondrial Complex Inhibition and Cell Cycle Arrest of CD8+ T Cells in Severe/Critical COVID-19. International Journal of Molecular Sciences, 24(16), 12780.

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Multiparametric Flow Cytometry Analysis

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Single-cell suspensions of splenocytes were mixed with PerCP anti-mouse CD3e, fluorescein isothiocyanate (FITC) anti-mouse CD4, allophycocyanin (APC)stained CD8 antibodies (BD Pharmingen), and phycoerythrin (PE)-stained CD45R/B220 antibodies. The mixtures were incubated at 4°C in the dark for 30 min. Subsequently, wash buffer was added, and the supernatant was discarded. Single cells were resuspended (flow buffer, 500 μL). The population of cells was identified using a FACSCalibur flow cytometer (BD Biosciences), following previously described protocols (Yadav et al., 2013) (link).

Liang X., Wang Z., Yang H., Luo X., Sun J., Yang M., Shi X., Yue X, & Zheng Y. (2022). Evaluation of allergenicity of cow milk treated with enzymatic hydrolysis through a mouse model of allergy. Journal of dairy science, 105(2).

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Mouse Spleen Cell Isolation and Characterization

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Single cell suspension was obtained from mouse spleen. Briefly, tissue was lysed with ammonium-chloride-potassium (eBioscience) and filtered to remove red blood cells. Following treatment with blocking antibody to restrain non-specific binding, suspended cells were incubated with fluorochrome-conjugated antibodies for 30 min at 4°C. Flow cytometry analysis was conducted using FACS Calibur (BD Biosciences) and data were processed using Flow Jo software (TreeStar, Inc.). CD3 antibody (Clone17A2, BioLegend), CD4 antibody (Clone RM4.5, BD Biosciences) and CD8 antibody (BD Biosciences) were used in this study.

Wang J., Chen X., Zhang L., Zheng Y., Qian J., Sun N., Ding X, & Cui B. (2022). Chick early amniotic fluid component improves heart function and protects against inflammation after myocardial infarction in mice. Frontiers in Cardiovascular Medicine, 9, 1042852.

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Evaluating Antigen-Presenting Capacity of CAFs

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20,000 sorted apCAFs from KPfC tumors were seeded in U-bottom 96-well plates and incubated with or without 25 μg/ml OVA peptide (OVA 323–339) (GenScript) in DMEM with 10% FBS for 4 hrs at 37°C. Spleens from 6-to-8-week-old OT II mice were harvested and CD4⁺ T cells were isolated using a MojoSort mouse CD4⁺ T cell isolation kit (Biolegend). apCAFs were washed 3 times and co-cultured with 50,000 CD4⁺ T cells in DMEM with 10% FBS for 18 hrs. CD4⁺ T cells were then collected and co-cultured with CD8⁺ T cells (1:1 ratio) from splenocytes of C57BL/6 mice that were isolated using MojoSort mouse CD8⁺ T cell isolation kit (Biolegend) and labeled with CellTrace CFSE dye (Thermo Fisher Scientific, 1 μM). After 72 hours, cells were stained with CD8 antibody (BD Biosciences, 1:100) and the percentage of proliferating CD8⁺ T cells was analyzed by CFSE signal with flow cytometry.

Huang H., Wang Z., Zhang Y., Pradhan R.N., Ganguly D., Chandra R., Murimwa G., Wright S., Gu X., Maddipati R., Müller S., Turley S.J, & Brekken R.A. (2022). Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer. Cancer cell, 40(6), 656-673.e7.

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RFP-Specific CD8+ T Cell Response

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C57BL/6 mice of age 6 weeks (Orient, Korea) were subcutaneously vaccinated with 10 μl of hFTN-RFP (10 μM), hFTN (10 μM), RFP (10 μM), PBS three times with 1-week interval. One week after the final vaccination, spleen was excised, and splenocytes were harvested from the spleen using cell strainer. Splenocytes were treated with red blood cell lysis buffer, incubated for 1 min at room temperature, and washed with PBS. Splenocytes (5 × 10⁶ cells/ml) from each vaccinated mouse were activated for 1 h with 1 μg/ml of RFP-derived peptide (S111 to I119 or SSLQDGCFI)35 (link) that acts as an epitope for RFP-primed response. Cells were stained with CD8 antibody (BD Biosciences, San Jose, CA). Then, intracellular IFN-γ staining was performed with the Cytofix/Cytoperm (BD Biosciences) using IFN-γ antibody (BD Biosciences). IFN-γ-secreting CD8⁺ T cells were characterized by fluorescence-activated cell sorting (FACS) analysis and were quantified using CELLQuest software.

Lee B.R., Ko H.K., Ryu J.H., Ahn K.Y., Lee Y.H., Oh S.J., Na J.H., Kim T.W., Byun Y., Kwon I.C., Kim K, & Lee J. (2016). Engineered Human Ferritin Nanoparticles for Direct Delivery of Tumor Antigens to Lymph Node and Cancer Immunotherapy. Scientific Reports, 6, 35182.

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Immunohistochemical Analysis of Tumor Samples

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Tumor tissues were rolled and fixed in formalin for 24 h, then embedded in paraffin. Sections of 5 μm were stained for H&E and mounted with Permount Mounting Medium (Thermo Fisher Scientific). For immunohistochemistry, paraffin tissue sections underwent antigen retrieval, blocking in 5% goat serum in PBS, and probed with Ki67 antibody (Cell Signaling, 1:250), OTC antibody (ThermoFisher 1:200), or CD8 antibody (BD Bioscience 1:200). Sections were washed twice with PBST and incubated with HRP conjugated anti-rabbit IgG (1:500, catalog 7074S, Cell Signaling Technology) for 1 h. Sections were then washed with PBST and stained with DAB substrate. After the color change, the reaction was stopped with distilled water and dehydration was completed before mounting with Permount Mounting Medium. For immunofluorescence OPAL staining, samples were initially dehydrated, then stained with the indicated fluorescent OPAL antibody, then analyzed by multispectral imaging and analyzed for spatial and morphological context. Histological scoring of dysplasia was done by a blinded pathologist.

Bell H.N., Huber A.K., Singhal R., Korimerla N., Rebernick R.J., Kumar R., El-derany M.O., Sajjakulnukit P., Das N.K., Kerk S.A., Solanki S., James J.G., Kim D., Zhang L., Chen B., Mehra R., Frankel T.L., Győrffy B., Fearon E.R., di Magliano M.P., Gonzalez F.J., Banerjee R., Wahl D.R., Lyssiotis C.A., Green M, & Shah Y.M. (2022). Microenvironmental Ammonia Enhances T Cell Exhaustion in Colorectal Cancer. Cell metabolism, 35(1), 134-149.e6.

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