Anti cd8

Manufactured by Miltenyi Biotec

Sourced in Germany

Anti-CD8 is a monoclonal antibody that binds to the CD8 receptor on the surface of T cells. It is a widely used tool in immunology research and flow cytometry applications to identify and study CD8+ T cells.

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Anti-CD4 and anti-CD8 magnetic beads APC‐conjugated anti‐CD8 REA734 mAb Anti-CD4 or anti-CD8-coated magnetic microbeads Anti-CD8 PE Anti-CD8 magnetic beads Anti-CD8 (clone BW135/80, Human anti-CD8 microbeads Anti-mouse-CD8-α (Ly-2) MicroBeads Anti-human CD8-APC Anti-CD4 and anti-CD8 MACS beads

8 protocols using anti cd8

Isolation and Activation of Immune Cells

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PBMCs were isolated from buffy coats from healthy donors (blood transfusion center at University Medical Center (UMC) Mainz, Germany) by Ficoll®-Hypaque (GE Healthcare) density gradient centrifugation. Monocytes were isolated using anti-CD14 microbeads (Miltenyi Biotec) and CD8 and CD4 cells were isolated using anti-CD8 and anti-CD4 microbeads, respectively (Miltenyi Biotec) according to the manufacturer’s protocol. Immature DCs (iDCs) were differentiated from CD14⁺ cells in RPMI 1640 GlutaMAX™, 50 IU/mL penicillin, 50 μg/mL streptomycin, and 10% (v/v) human AB serum (one lambda) (denoted huRPMI), supplemented with 1000 IU/mL recombinant human (rh) GM-CSF and 1000 IU/mL rh IL-4 (Miltenyi Biotec) twice in 5 days. For T-cell activation, 2 × 10⁶ / ml of isolated T-cells were cultivated in 24 well tissue culture plates precoated with 2 μg/ml anti-CD3 antibody (clone OKT3; Bioxcell) in huRPMI supplemented with 100 IU/mL IL-2 for 2 to 3 days. Cells were then harvested, washed and resuspended in huRPMI with 50 IU/mL IL-2 and either rested or immediately submitted to further processing steps.

Alishah K., Birtel M., Masoumi E., Jafarzadeh L., Mirzaee H.R., Hadjati J., Voss R.H., Diken M, & Asad S. (2021). CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro. Journal of Translational Medicine, 19, 482.

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Adoptive Transfer of Immunized Cells

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Cell suspensions from spleens and lymph nodes of mice immunized or tolerized with Cas antigen or Cas-coupled erythrocytes, respectively, were positively selected on MiniMACS MS columns with microbeads coated with monoclonal antibodies anti-CD4 or anti-CD8 according to manufacturer’s procedures (Miltenyi Biotec, Bergisch Gladbach, Germany). Selected viable cells were suspended in DPBS and intravenously administered to naive mice in adoptive transfer (see above).

Wąsik M., Nazimek K., Nowak B., Askenase P.W, & Bryniarski K. (2019). Delayed-Type Hypersensitivity Underlying Casein Allergy Is Suppressed by Extracellular Vesicles Carrying miRNA-150. Nutrients, 11(4), 907.

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Isolating Tumor-Infiltrating T Cells

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To isolate T cells from tumors for cytospin preparation or western blot, WT mice bearing B16F10 tumors were infected with LCMVa or RPMI control. 8 days post-infection, tumors were harvested and homogenized by mechanical mincing and incubation in Type II collagenase (Worthington) in HBSS for one hour at 37 degrees C with shaking. Tumor homogenates were then passed through a 100 μm strainer to obtain a single cell suspension, and CD8 or CD45 cells were isolated using anti-CD8 or anti-CD45 beads for positive selection in magnetic columns per manufacturer’s protocol (Miltenyi). For western blots, CD8⁺ cells were then incubated for 4 hours with Brefeldin-A in splenocyte medium before cells were lysed using RIPA. For cytospins, 150,000 CD45⁺ cells per slide were immediately added to slides. Slides were then fixed in 4% paraformaldehyde and immunofluorescent staining was performed with TSP-1 and CD3 antibodies as described above.

Schadler K.L., Crosby E.J., Zhou A.Y., Bhang D.H., Braunstein L., Baek K.H., Crawford D., Crawford A., Angelosanto J., Wherry E.J, & Ryeom S. (2014). Immunosurveillance by anti-angiogenesis: tumor growth arrest by T cell-derived thrombospondin-1. Cancer research, 74(8), 2171-2181.

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Flow Cytometry Analysis of Immune Cells

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Monoclonal antibodies were purchased from Thermo Fisher Scientific eBioscience or BD Bioscience. Clones used were: anti-CD4 (RM4-5, dilution 1:500), anti-CD8α (53–6.7 dilution 1:500), anti-TCRβ (H57-597, dilution 1:250), anti-HSA (M1/69, dilution 1:500), anti-CD69 (H1.2F3, dilution 1:250), anti-CD44 (IM7, dilution 1:500), anti-CD62L (MEL-14, dilution 1:500), anti-CD19 (ID3, dilution 1:300), anti-CD127 (SB/199, dilution 1:250), anti-Caspase-3 (C92-605, 10 μl of Ab per test). Cell proliferation dye E450 was obtained from Thermo Fisher Scientific eBioscience. After staining with antibodies and DAPI (Thermo Fisher Scientific), cells were analyzed with an LSRII flow cytometer (BD Biosciences) or sorted with an Aria II (BD Biosciences). Post sort sample purity was >98%. In most cases, anti-CD4, anti-CD8, anti-PE, and anti-B220 magnetic beads (Miltenyi) were used for enrichment and depletion on MACS columns (Miltenyi) before sorting. Flow cytometry data were analyzed using Flowjo software (Tree Star).

Issuree P.D., Day K., Au C., Raviram R., Zappile P., Skok J.A., Xue H.H., Myers R.M, & Littman D.R. (2018). Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development. Nature Communications, 9, 3594.

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Comprehensive T-cell Phenotyping by Flow Cytometry

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Cellular composition, CD4/CD8 ratio and CAR-expression was determined by flow cytometric analysis after staining with one or several of the following antibodies and reagents: anti-CD45, anti-CD4, anti-CD3, anti-CD16, anti-CD56, anti-CD19, anti-CD14, anti-CD8, anti-CD45RO, anti-CD95, anti-CD62L and 7-AAD (all Miltenyi Biotec). CAR-modified T cells were identified using the EGFRt marker and staining with an anti-EGFRt antibody (clone: C225, ImClone Systems) conjugated in-house to AlexaFluor 647. Data were acquired on a MACSQuant Analyzer 10 and analyzed using MACSQuantify software (both Miltenyi Biotec).

Lock D., Monjezi R., Brandes C., Bates S., Lennartz S., Teppert K., Gehrke L., Karasakalidou-Seidt R., Lukic T., Schmeer M., Schleef M., Werchau N., Eyrich M., Assenmacher M., Kaiser A., Prommersberger S., Schaser T, & Hudecek M. (2022). Automated, scaled, transposon-based production of CAR T cells. Journal for Immunotherapy of Cancer, 10(9), e005189.

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Flow Cytometry Immunophenotyping Assay

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The following FITC- or PE-conjugated mAbs were purchased from BD Pharmingen (San Diego, CA), eBioscience (San Diego, CA), R&D Systems (Minneapolis, MN), Abcam (Bristol, UK) or Miltenyi Biotec (Bergisch Gladbach, Germany): anti-HLA class II (clone TU39, Mouse IgG2a), anti-HLA class I (clone G46-2.6, mouse IgG1), anti-CD80 (clone L307.4, mouse IgG1), anti-CD83 (clone HB15e, mouse IgG1), anti-CD86 (clone FUN-1, mouse IgG1), anti-CD40 (clone 5C3, mouse IgG1), and anti-CD8 (clone T8, mouse IgG1). Mouse IgG2a (clone G155-178), mouse IgG2b (clone 27–35), mouse IgG1 (clone MOPC-21), affinity purified mouse IgM (eBioscience), and rat IgG2a (clone eBR2a) were used as isotype-matched controls. The cell samples were treated with an Fc-receptor-blocking reagent (Miltenyi Biotec) for 10 min, stained with the fluorochrome-conjugated mAb for 30 min, and washed 3 times with PBS containing 2% FCS. The stained cell samples were analyzed on a FACScan (BD Biosciences, Bedford, MA) flow cytometer.

Imamura Y., Haruta M., Tomita Y., Matsumura K., Ikeda T., Yuno A., Hirayama M., Nakayama H., Mizuta H., Nishimura Y, & Senju S. (2016). Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology. PLoS ONE, 11(4), e0152384.

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Intestinal Lymphocyte Isolation and Characterization

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Intestinal cells were dissociated using the lamina propria dissociation kit (Miltenyi Biotec) in combination with gentleMACS^™ Octo Dissociator with Heaters (Miltenyi Biotec). Percoll gradient separation was performed using 40% Percoll underlaid with 80% Percoll and lymphocytes at interphase were collected. Lymphocytes were stimulated using the Cell Activation Cocktail with Brefeldin A (Biolegend). The following stains were used for extracellular staining: LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen; Cat#L34955), Rat Anti-Mouse CD16/CD32 (BD; Cat#553141, Clone: 2.4G2), anti-CD3 (Miltenyi Biotec; Cat#130-102-943, Clone: REA641), and anti-CD4 (Invitrogen; Cat#25-0041-82, Clone: GK1.5), and anti-CD8 (Miltenyi Biotec; Cat#130-120-806, REA601). Foxp3/Transcription Factor Staining Buffer Set (Invitrogen; Cat#00-5523-00) was used for permeabilizing cells for intracellular staining. The following stains were used for intracellular staining: anti-IL17 (Miltenyi Biotec; Cat#130-102-262, Clone: TC11-18H10), anti-IL4 (Miltenyi Biotec; Cat#130-102-435, Clone: BVD4-1D11), and anti-IFN-y (Miltenyi Biotec; Cat#130-102-388, Clone: AN.18.17.24), examined by LSR II cell analyzer (Becton, Dickinson and Company, USA) and analyzed by FlowJo v10.6 software (Becton, Dickinson and Company, USA).

ter Haar E., Harrison S.C, & Kirchhausen T. (2000). Peptide-in-groove interactions link target proteins to the β-propeller of clathrin. Proceedings of the National Academy of Sciences of the United States of America, 97(3), 1096-1100.

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Adoptive T Cell Transfer Assay

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Single-cell suspensions from spleens were prepared by mechanical disruption of the organ and subjected to hypotonic red blood cell lysis. For in vivo proliferation, splenocytes were labeled using CFSE (Molecular Probes) according to the manufacturer’s protocol, and 10⁷ cells (corresponding to 1 × 10⁶ CD8⁺ TCR transgenic T cells) were transferred intravenously (i.v.) into recipient mice. For in vivo protection and co-transfer study, CD8⁺ T cells or CD3⁺ cells from spleens were obtained using anti-CD8 or anti-CD3 MACS beads (Miltenyi Biotec). For assessment of in vivo protection, recipients received 10⁶ cells, while 10⁵ and 10⁴ cells were transferred to plt/plt mice in separate or competitive transfer assays (respectively). At twelve hours post adoptive transfer, recipient mice were infected with MHV A59.

Cupovic J., Onder L., Gil-Cruz C., Weiler E., Caviezel-Firner S., Perez-Shibayama C., Rülicke T., Bechmann I, & Ludewig B. (2016). Central Nervous System Stromal Cells Control Local CD8+ T Cell Responses during Virus-Induced Neuroinflammation. Immunity, 44(3), 622-633.

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