Apc anti cd8a

Manufactured by Thermo Fisher Scientific

Sourced in United States

APC-anti-CD8a is a fluorochrome-conjugated antibody that binds to the CD8a surface antigen, which is expressed on a subset of T cells. This antibody can be used in flow cytometry applications to identify and enumerate CD8+ T cells.

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Anti-CD8a (18 citations) CD8a-APC (14 citations) APC anti-mouse CD8a (6 citations) Anti-CD8a-APC-eFluor 780 (clone RPA-T8; (3 citations) Anti-Rat CD8a APC (3 citations) Anti CD8a-APC (53-6.7) (2 citations)

9 protocols using apc anti cd8a

Multiparametric Flow Cytometry Analysis

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Liver mononuclear cells (MNCs) were purified by a 40%/70% percoll gradient^{48 (link)} and stained with anti-CD3-PE, anti-CD4-PerCP/Cy5.5, anti-CD8a-APC, or anti-CD25-FITC (eBiosciences) for 30 min at 4 °C in staining buffer (PBS, 3% FCS). For detection of intracellular proteins, cells were first stimulated with PMA (Sigma) and inomycin (Sigma) for 4 h and stopped by addition of Brefeldin A (Sigma). After cell surface staining, cells were fixed and permeabilized (eBiosciences), and then stained respectively with anti-IFNγ-FITC, anti-IL-17 A-PE, or anti-Foxp3-PE. For detection of surface expression of IL-17RA in cloned MSCs, cells were stained with anti-IL-17RA-PE (eBiosciences). All samples were analyzed by flow cytometry on a FACS Calibur flow cytometer (Becton Dickinson).

Han X., Yang Q., Lin L., Xu C., Zheng C., Chen X., Han Y., Li M., Cao W., Cao K., Chen Q., Xu G., Zhang Y., Zhang J., Schneider R.J., Qian Y., Wang Y., Brewer G, & Shi Y. (2014). Interleukin-17 enhances immunosuppression by mesenchymal stem cells. Cell Death and Differentiation, 21(11), 1758-1768.

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Tumor-Infiltrating T-Cell Analysis

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At the time of sacrifice, the tumors and spleens were harvested from the mice. Single-cell suspensions of splenocytes were stained with anti-CD3-FITC (cat. no. 11-0032-82; eBioscience, San Diego, USA), anti-CD4-PerCP/Cy5.5 (cat. no. 100540; BioLegend) and anti-CD8a-APC (cat. no. 17-0081-83; eBioscience) at 4 °C for 20 min. The single-cell suspensions of tumors were stained with anti-CD45-R-PE (cat. no. 147711; BioLegend), anti-CD3-FITC, anti-CD8a-APC and anti-IFN-γ-Bv421 (cat. no. 505830; BioLegend) at 4 °C for 20 min. The cells were analyzed by an Attune NxT Flow Cytometer.

Ren S., Wang X, & Jin G. (2022). Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity. International Journal of Biological Sciences, 18(1), 166-179.

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Immune Cell Profiling of Murine Tumors

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Orthotopic Met-1 or AT3 tumours were isolated, dissociated into single cell suspension, and incubated for 15 min with anti-mouse CD16/CD32 to reduce non-specific staining. Infiltration of immune cells into tumours was analysed by staining with the following anti-mouse antibodies: CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), CD45-BV570 (Biolegend, 103136, diluted 1:100), CD3e-FITC (eBioscience, 14-0031-82, diluted 1:100), CD4-APC/Cy7 (Biolegend, 100414, diluted 1:100), anti-CD8a-APC (eBioscience, 17-0081-82, diluted 1:100), CD11b-PerCP/Cy5.5 (eBioscience; 45-0112-82, diluted 1:100), F4/80-APC (eBioscience, 17-4801-82, diluted 1:50), Ly-6G/Ly-6C (Gr-1)-APC/Cy7 (Biolegend, 108424, diluted 1:100), Ly-6G-APC (Biolegend, 127614, diluted 1:200), Ly-6C-FITC (Biolegend, 128006, diluted 1:200), Ly-6C-BV605 (Biolegend, 128036, diluted 1:200). DAPI was used to exclude dead cells. Acquisition was performed with Beckman-Coulter Gallios flow cytometer and data analysis was done with FlowJo software (version X.0.7).

Ershaid N., Sharon Y., Doron H., Raz Y., Shani O., Cohen N., Monteran L., Leider-Trejo L., Ben-Shmuel A., Yassin M., Gerlic M., Ben-Baruch A., Pasmanik-Chor M., Apte R, & Erez N. (2019). NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis. Nature Communications, 10, 4375.

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Isolation and Characterization of Immune Cells from Mouse Brain

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Mouse brains without the cerebellum were coarsely chopped and incubated in a 0.01% collagenase (Sigma–Aldrich, #C0130) solution in PBS at 37 °C for 40 min under agitation. Samples were passed through a 70 μm filter in a total volume of 20 mL PBS. Cell suspension was mixed with a Percoll solution (30% Percoll, GE Healthcare #17089101, 3.33% PBS 10x, and 66,6% PBS 1 ×) and centrifuged for 30 min at 1500 × g. After centrifugation, the supernatant was discarded. Cells forming the pellet were washed twice with PBS, resuspended in PBS supplemented with 2% FCS and incubated for 30 min on ice with fluorescence-conjugated antibodies (1:100 dilution; anti-CD45 FITC, Tonbo Biosciences clone 30-F11; anti-CD11b PECy7, eBiosciences clone M1/70; anti-CD4 PE, Invitrogen #12-0042082; anti-CD8a APC, eBiosciences clone 53-6.7). Samples were washed twice with PBS before cytometry. Acquisition was performed on a MoFlo XDP FACS equipment (Beckman Coulter Inc., Brea, CA). Sorted cells were CD45^hiCD4⁺ (T CD4 lymphocytes), CD45^hiCD8⁺ (T CD8 lymphocytes), CD45^hiCD11b^hi (activated microglia and/or infiltrating macrophages) and CD45^intCD11b^lo (quiescent microglia). Data were analyzed with FlowJo vX software (Tree Star Inc., Ashland, OR).

Figueiredo C.P., Barros-Aragão F.G., Neris R.L., Frost P.S., Soares C., Souza I.N., Zeidler J.D., Zamberlan D.C., de Sousa V.L., Souza A.S., Guimarães A.L., Bellio M., Marcondes de Souza J., Alves-Leon S.V., Neves G.A., Paula-Neto H.A., Castro N.G., De Felice F.G., Assunção-Miranda I., Clarke J.R., Da Poian A.T, & Ferreira S.T. (2019). Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice. Nature Communications, 10, 3890.

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T Cell Phenotypic and Functional Analyses

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The following reagents and fluorochrome-conjugated monoclonal antibodies were used in this study for T cell phenotypic and functional analyses: PKH26 membrane dye (Sigma), APC- anti-CD4, APC-anti-CD8a, PE-anti-CD4, PE-anti-CD25, FITC or APC-anti-TNFα, PE-anti-ICOS-L FITC-Annexin-V (all from eBioscience), FITC-anti-CXCR4, APC-anti-CXCR7, PE-anti-hCCR4 (all from R&D Systems), and FITC-anti-CD279/PD-1 (Biolegend, San Diego, CA). Intracellular expression of TNFα was detected following fixation and permeabilization (fixation/permeabilization buffer concentrate, Cat. No. 00-5123-43, diluent, Cat. No. 00-5223-56, and permeabilization buffer, Cat. No. 00-8333-56, all from eBioscience). All samples were analyzed with a FACSCalibur flow cytometer, using CellQuest software (BD Biosciences). For analysis of tumor ascites CD14⁺ cell phenotype, the following monoclonal antibodies were used: FITC-anti-CD14, PE-anti-CD11b (both from R & D Systems), PE-anti-CD11c, PE-anti-B7-H1, PerCP-anti-CD14 (all from BD Biosciences), FITC-anti-CD279/PD-1, FITC-anti-CD83, FITC-anti-CD80, PE-anti-CD86, APC-anti-CD14, and PE-IL-1β (all from eBioscience). Intracellular expression of IL-1β was assayed following overnight incubation of CD14⁺ cells in the presence of Brefeldin A, followed by fixation and permeabilization as described above.

Goyne H.E., Stone P.J., Burnett A.F, & Cannon M.J. (2014). Ovarian tumor ascites CD14+ cells suppress dendritic cell-activated CD4+ T cell responses through IL-10 secretion and indoleamine 2,3-dioxygenase. Journal of immunotherapy (Hagerstown, Md. : 1997), 37(3), 163-169.

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Multiparametric Flow Cytometry for Immune Cell Profiling

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The following anti-mouse antibodies were from eBioscience: FITC-anti-F4/80, APC-anti-CD80, PE-Cy7-anti-CD86, PE-anti-MHC-II, PE-anti-CD11b, FITC-anti-Gr-1, APC-Cy7-anti-CD3, PerCP-Cyanine5.5-anti-CD4, APC-anti-CD8a, PE-anti-IFN-γ, and FITC-anti-IL-17. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml, Enzo Life Sciences) and ionomycin (1 nM, Enzo Life Sciences) in the presence of brefeldin A (1 mg/ml, Enzo Life Sciences) for 4–5 h. Surface staining was performed in FACS buffer (0.5% BSA, 2 mM EDTA, 0.02% sodium azide in PBS) in the presence of Fc receptor blocking antibody (BioLegend) for 20 min at 4°C. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature followed by permeabilization (0.1% BSA, 0.5% saponin in PBS) or with the BD Cytofix/Cytoperm Fixation and Permeabilization Kit. Cytokine staining was performed in permeabilization buffer for 20 min at 4°C. Data acquisition and analysis were performed using BD LSRFortessa X-20 (BD Bioscience) and FlowJo software (Tree Star, Ashland, OR, USA) respectively.

He W., Hu S., Du X., Wen Q., Zhong X.P., Zhou X., Zhou C., Xiong W., Gao Y., Zhang S., Wang R., Yang J, & Ma L. (2018). Vitamin B5 Reduces Bacterial Growth via Regulating Innate Immunity and Adaptive Immunity in Mice Infected with Mycobacterium tuberculosis. Frontiers in Immunology, 9, 365.

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Intracellular Cytokine Detection Protocol

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For intracellular cytokine detection, isolated cells were cultured in 20 μg/ml of M. tuberculosis lysate or PMA/ionomycin for 1.5 h before 10 μg/ml Brefeldin A (eBioscience, USA) was added to the culture for 3.5 h more. For surface staining, lymphocytes were harvested, washed and stained for 30 min on ice with mixtures of fluorescently conjugated mAbs or isotype-matched controls. mAbs of mice were as follows: APC-Cy7-anti-CD3, PE-Cy7-anti-CD4, APC-anti-CD8a, PE-anti-CD45.1, Alexa Fluor 700-anti-CD45.2, PE-anti-CD25, PerCP-cy5.5-anti-CD69, PE-anti-CD44, FITC-anti-CD62L, PE-cy7-anti-Gr-1, PE-anti-CD11b, APC-ant-CD86, Alexa Fluor 700-anti-MHC-II and PerCP-cy5.5-anti-CD206 (eBioscience). For intracellular staining, the cells were incubated 20 min in IC Fixation buffer (eBioscience), followed by permeabilization buffer (eBioscience) and 1 h of incubation with appropriate mAbs of mice: PE-anti-IFN-γ, FITC-anti-IL-17, PerCP-cy5.5-anti-IL-4, FITC-anti-IL-2 and FITC-anti-Foxp3. Cell phenotype was analyzed by flow cytometry on a flow cytometer (BD LSR II) (BD Biosciences, USA). Data were acquired as the fraction of labeled cells within a live-cell gate and analyzed using FlowJo software (Tree Star). All gates were set on the basis of isotype-matched control antibodies.

Hu S., Du X., Huang Y., Fu Y., Yang Y., Zhan X., He W., Wen Q., Zhou X., Zhou C., Zhong X.P., Yang J., Xiong W., Wang R., Gao Y, & Ma L. (2018). NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection. PLoS Pathogens, 14(8), e1007266.

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CD4+ Cell Depletion and IL-17A Neutralization

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CD4⁺ cells were depleted using anti-CD4 monoclonal depletion antibody (clone: GK1.5) or matching isotype control (Rat IgG2B). IL-17A neutralization was performed using anti-IL-17A monoclonal antibody (clone: 17F3) or matching isotype control (Mouse IgG1). Antibodies were administered intraperitoneally, 500μg per day every 3 days for a total of 3 doses. Efficacy of CD4⁺ depletion was determined in colonic lamina propria and spleen by flow cytometry. For intestinal lamina propria lymphocytes isolation, tissue pieces were washed with cold PBS and incubated in RPMI with 1 mg/ml Collagenase/Dispase for 30 min at 37°C with shaking at 200 rpm. Splenocytes were disrupted into single cell suspension by passing the organ through 70 μm filter and RBCs were lysed in ACK lysis buffer (Invitrogen) for 3 min. Cells were stained using the following monoclonal fluorescence-conjugated antibodies: BUV395 anti-CD45.2 (Clone: 104, BD Biosciences), APC-eFluor 780 anti-CD4 (Clone: RM4–5, eBioscience), and APC anti-CD8a (Clone: 53–6.7, eBioscience). All antibodies were diluted in FACS buffer (2% FBS, 0.01 Sodium Azide, PBS). Dead cells were gated out by using the Fixable Violet Dead Cell Stain Kit (Invitrogen). Samples were acquired on the BD LSRFortessa (BD Biosciences) and analyzed with FlowJo Software (Treestar).

Woo V., Eshleman E.M., Hashimoto-Hill S., Whitt J., Wu S.E., Engleman L., Rice T., Karns R., Qualls J.E., Haslam D.B., Vallance B.A, & Alenghat T. (2021). Commensal segmented filamentous bacteria-derived retinoic acid primes host defense to intestinal infection. Cell host & microbe, 29(12), 1744-1756.e5.

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CD4+ Cell Depletion and Lamina Propria Isolation

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CD4 + cells were depleted using anti-CD4 monoclonal depletion antibody (clone: GK1.5) or matching isotype control (Rat IgG2B) administered intraperitoneally, 500µg per day every 3 days for a total of 3 doses. Efficacy of CD4 + depletion was determined in colonic lamina propria and spleen by flow cytometry. For intestinal lamina propria lymphocytes isolation, tissue pieces were washed with cold PBS and incubated in RPMI with 1 mg/ml Collagenase/Dispase for 30 min at 37˙C with shaking at 200 rpm. Splenocytes were disrupted into single cell suspension by passing the organ through 70 µm filter and RBCs were lysed in ACK lysis buffer (Invitrogen) for 3 min. Cells were stained using the following monoclonal fluorescence-conjugated antibodies: BUV395 anti-CD45.2 (Clone: 104, BD Biosciences), APC-eFluor 780 anti-CD4 (Clone: RM4-5, eBioscience), and APC anti-CD8a (Clone: 53-6.7, eBioscience). All antibodies were diluted in FACS buffer (2% FBS, 0.01 Sodium Azide, PBS). Dead cells were gated out by using the Fixable Violet Dead Cell Stain Kit (Invitrogen). Samples were acquired on the BD LSRFortessa (BD Biosciences) and analyzed with FlowJo Software (Treestar).

Woo V., Eshleman E.M., Whitt J., Hashimoto-Hill S., Wu S., Engleman L., Rice T., Karns R., Vallance B.A., & Alenghat T. (2021). Commensal bacterial-derived retinoic acid primes host defense to intestinal infection.

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