Anti cd8 percp

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-CD8-PerCP is a fluorescent antibody product used in flow cytometry applications. It binds to the CD8 protein expressed on the surface of certain T cells. The PerCP fluorochrome attached to the antibody allows for the detection and quantification of CD8+ T cells in a sample.

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Close spelling variants of this product

Anti-CD8 (106 citations) CD8-PerCP (13 citations) Anti-CD8-PerCP-Cy5.5 (13 citations) PerCP-Cy5.5 anti-mouse CD8 (4 citations) PerCP-Cy5.5 anti-CD8 (3 citations) Anti-CD8-PerCP-Cy5.5 (53–6.7, (3 citations) Anti-CD8 (PerCP 53-6.7, (2 citations) PerCP-Cy5.5-labeled anti-CD8 (2 citations)

7 protocols using anti cd8 percp

Isolation and Characterization of Murine Immune Cells

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Mononuclear cells were isolated from spleens and MLN by gentle extrusion of the tissue through a 50 μm-mesh Nylon cell strainer (BD). Cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% of fetal calf serum (FCS), 2 mM L-glutamine, 50 U/mg penicillin, and 50 U/mg streptomycin (Lonza, Levallois-Perret, France). Erythrocytes were lysed with red blood-cell lysing buffer (Sigma–Aldrich).
For flow cytometry analysis, aliquots of 10⁶–10⁷ cells per sample were pre-incubated with purified anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) and then labeled with anti-CD4-FITC, anti-CD3e-PE, and anti-CD8-PerCP (all from eBioscience) according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry (Accuri, BDbioscience) with CFlow Sampler software (BD).
For stimulation experiments, 2 × 10⁵ cells per well were cultured for 48 h (37°C, 10% CO₂) in DMEM medium in P24 plates pre-coated with anti-CD3/CD28 antibodies (4 μg/mL each; eBioscience) or phorbol 12-myristate 13-acetate (PMA)/ionomycin (cell stimulation cocktail, 1×, ebioscience). Culture supernatant was frozen at −80°C until processing.

Martín R., Laval L., Chain F., Miquel S., Natividad J., Cherbuy C., Sokol H., Verdu E.F., van Hylckama Vlieg J., Bermudez-Humaran L.G., Smokvina T, & Langella P. (2016). Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice. Frontiers in Microbiology, 7, 608.

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Surface Staining of Peripheral Blood Cells

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For surface staining, 200 ul of anticoagulated peripheral blood were stained during 30 min at room temperature with a combination of the following Abs: anti-CD19 PerCPCy5.5 (HIB19, BD), anti-CD19 APCCy7 (HIB19, Biolegend), anti-CD24 FITC (ML5, BD), anti-CD38 APC (HIT2, BD), anti-PD-L1 PECy7 (MIH1, BD), control isotype (MOPC-21, BD), anti-CD4 FITC (13B8.2, Beckman Coulter, Brea, CA), anti-CD8 PerCP, and anti-CD8 APC (RPA-T8, eBioscience. After staining, red blood cells were lysed with 5 ml of cold lysing buffer (NH4Cl 0.15M, KHCO3 10 mM, Na2EDTA 0.1 mM, in distilled water) during 20 min at 4°C. Then, samples were centrifuged, washed with PBS and resuspended in 2%FBS-PBS and acquired on a BD FACSCanto II Flow Cytometry. The analysis was performed using FlowJo software (version X).

Zacca E.R., Onofrio L.I., Acosta C.D., Ferrero P.V., Alonso S.M., Ramello M.C., Mussano E., Onetti L., Cadile I.I., Stancich M.I., Taboada Bonfanti M.C., Montes C.L., Acosta Rodríguez E.V, & Gruppi A. (2018). PD-L1+ Regulatory B Cells Are Significantly Decreased in Rheumatoid Arthritis Patients and Increase After Successful Treatment. Frontiers in Immunology, 9, 2241.

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Flow Cytometry Analysis of Lymph Node Cells

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Popliteal lymph nodes were removed and macerated as described earlier. Cells were quantified by light microscope using trypan blue and then plated at 1 × 10⁶ cells/well in a 96-well plate. Staining of intracellular and extracellular markers was performed following manufacturer's instructions. Briefly, cells were re-stimulated ex vivo for 4 h with phorbol 12-myristate 13-acetate (PMA; 20 ng/mL) plus ionomycin (1 μg/mL) in the presence of a Golgi complex inhibitor (brefeldinA) for intracellular cytokine analysis. Extracellular markers were stained, the cells were fixed and permeabilized to enable intracellular staining. Cells from lymph nodes of infected and control mice were phenotyped according to the criteria described by Cossarizza et al. (10 (link)). The antibodies used in this work were: anti-CD3-Pacific Blue (eBioscience) (1:200), anti-CD4-PeCy7 (eBioscience) (1:200), anti-B220-PercP 5.5 (eBiosciences) (1:200), anti-TCRγδ-FITC (eBioscience) (1:200) and anti-CD8-PercP (eBioscience) (1:200) for the extracellular markers, and anti-IL-17-APC (eBiosciences) (1:100) and anti-IFN-γ-APC (eBiosciences) (1:100) for the intracellular markers. Analysis was performed in the FlowJo software. Gate strategy for IFN-γ-producing cells on Supplementary Figure 7 and for IL-17- producing cells on Supplementary Figure 8.

dos-Santos J.S., Firmino-Cruz L., Ramos T.D., da Fonseca-Martins A.M., Oliveira-Maciel D., De-Medeiros J.V., Chaves S.P., Gomes D.C, & de Matos Guedes H.L. (2019). Characterization of Sv129 Mice as a Susceptible Model to Leishmania amazonensis. Frontiers in Medicine, 6, 100.

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Popliteal Lymph Node Multiparametric Analysis

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Popliteal lymph nodes were removed and macerated as described earlier. Briefly, cells were quantified by light microscope using trypan blue and then plated at 1 × 10⁶ cells/well in a 96-well plate. Staining of intracellular and extracellular markers was performed following the manufacturer’s instructions. The cells were re-stimulated ex vivo for 4 h with phorbol 12-myristate 13-acetate (PMA; 20 ng/mL) plus ionomycin (1 μg/mL) in the presence of a Golgi complex inhibitor (brefeldinA, St. Louis, MI, USA) for intracellular cytokine analysis. Extracellular markers were stained, and the cells were fixed and permeabilized to enable intracellular staining. The antibodies used in this work were as follows: anti-CD3-Pacific Blue (eBioscience, San Diego, CA, USA) (1:200), anti-CD4-PECy7 (eBioscience, San Diego, California, USA) (1:200), and anti-CD8-PercP (eBioscience, San Diego, CA, USA) (1:200) for the extracellular markers, and IFN-γ-APC (eBiosciences, San Diego, CA, USA) (1:100) for the intracellular marker. Analysis was performed in the FlowJo software (Ashland, OR, USA).

Oliveira-Maciel D., dos-Santos J.S., Oliveira-Silva G., de Mello M.F., da Fonseca-Martins A.M., Carneiro M.P., Ramos T.D., Firmino-Cruz L., Gomes D.C., Rossi-Bergmann B, & de Matos Guedes H.L. (2021). MPLA and AddaVax® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis. Microorganisms, 9(6), 1272.

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Multiparameter Surface Marker Profiling

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The following antibodies were used for surface marker staining: Anti-human goat F(ab) IgG (H+L) PE antibody (cat. no. 109-116-088) from Dianova; anti-CD3-PE eFluor 610 (cat. no. 61-0038-42), and anti-CD4-Alexa Fluor 700 (cat. no. 560049-42) from eBioscience, San Diego; anti-CD8-PerCP (cat. no. 344708), anti-CD10-APC (cat. no. 312210), anti-human CD223-APC (LAG-3, Cat. 369212), anti-PD-1-Alexa Fluor 488 (cat. no. 329935), anti-human CD366 (Tim-3, Cat. 345007) all from Biolegend; anti-CD3-V500 (cat. no. 561416) from BD Biosciences.

Wang S., Sellner L., Wang L., Sauer T., Neuber B., Gong W., Stock S., Ni M., Yao H., Kleist C., Schmitt A., Müller-Tidow C., Schmitt M, & Schubert M.L. (2021). Combining selective inhibitors of nuclear export (SINEs) with chimeric antigen receptor (CAR) T cells for CD19-positive malignancies. Oncology Reports, 46(2), 170.

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Virus-Induced Lung CD8+ T Cell Response

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To look at virus induced lung resident memory CD8+ T cell response, lungs at 27 days after the initial challenge were analyzed from each group (n = 5 mice/group but n = 4 mice/ 3X TIV NC). Before the lungs were harvested, anti-CD45 antibody (AF700 from eBioscience; 3 μg/mouse in 100 μl PBS) were given retro-orbitally after mice were knocked down with pentobarbital. Immediately after, lungs were harvested and single-cell suspension in 1X PBS were made by forcing lungs through 70 um cell strainer. After lung cell suspensions were treated with red blood cell lysis buffer, they were stained with anti-CD44-PECy7, anti-CD3-FITC, anti-CD8-PerCP, anti-CD103-APC, anti-CD69-PE-CF594, and viability dye-e450 (all eBioscience) along with Fc receptor blocking anti-CD16/CD32 (BD).

Choi A., Ibañez L.I., Strohmeier S., Krammer F., García-Sastre A, & Schotsaert M. (2020). Non-sterilizing, Infection-Permissive Vaccination With Inactivated Influenza Virus Vaccine Reshapes Subsequent Virus Infection-Induced Protective Heterosubtypic Immunity From Cellular to Humoral Cross-Reactive Immune Responses. Frontiers in Immunology, 11, 1166.

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CFSE Proliferation and Apoptosis Assay

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Carboxylfluorescein succinimidyl ester (CFSE) labeling was used to detect the proliferation of CD3 + T cells. Briefly, 1.3x10 6 CD3+ T cells were co-cultured with MDSCs at ratios of 1:0, 1:1, 3:1 and 10:1 in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, USA) and stimulated with anti-CD3 and CD28 monoclonal antibodies (Sigma-Aldrich). All CD3+ T cells were seeded into a 96-well plate (Costar, Lowell, MA, USA) in the presence or absence of MDSCs, as mentioned above. The cells were harvested following culturing for 5 days and stained with anti-CD4-APC and anti-CD8-PerCp. As the CFSE signal was diluted with each cell division, cells exhibiting low fluorescence intensity of CFSE were considered to have proliferated. IFN-γ in the supernatant was measured using an ELISA kit (RapidBio Laboratory, Calabasas, CA, USA), according to the manufacturer's instructions.
Apoptosis assay. The CD3+ T cells were co-cultured with MDSCs at the ratios mentioned above and treated with anti-CD3 and CD28 monoclonal antibodies. Following incubation for 48 h, the cells were collected and stained with annexin-V-FITC, 7-amino-actinomycin D (7-AAD) (eBioscience, San Diego, CA, USA), anti-CD4-APC and anti-CD8-PerCp to analyze the apoptosis of the CD4 and CD8 cells.

Li H., Dai F., Peng Q., Gan H., Zheng J., Xia Y, & Zhang W. (2015). Myeloid-derived suppressor cells suppress CD4⁺ and CD8⁺ T cell responses in autoimmune hepatitis. Molecular medicine reports, 12(3).

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