Percp cy5.5 cd8a

Manufactured by BioLegend

Sourced in United States

PerCP/Cy5.5-CD8a is a fluorescent-conjugated antibody that binds to the CD8a surface marker. It is used for the detection and analysis of CD8+ T cells in flow cytometry applications.

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4 protocols using percp cy5.5 cd8a

Quantifying Cytokine Levels in PBLs

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IL-4 and IFN-γ ready-to-use sandwich enzyme-linked immunosorbent assay (ELISA) kits were
obtained from Excell Biotech Co., Ltd. (Taicang, China). FACS lysing solution was obtained
from BD Biosciences (Gliwice, Poland). Fluorescently-labeled anti-mouse monoclonal
antibodies (FITC-CD3, PE-CD4, PerCP/Cy5.5-CD8a, and PE-CD19) were obtained from BioLegend
(San Diego, CA, USA). APS (>70%) was obtained from Shanghai Macklin Biochemical Co.,
Ltd. (Shanghai, China). All chemical reagents were of analytical purity and were purchased
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All material used in this
study was endotoxin-free, including not only the PBLs but also all biological and
synthetic substrates.

TANG J., WEI X., LI Y., JIANG L., FENG T., ZHU H., LI M., CHEN G., YU X., ZHANG J, & ZHANG X. (2020). Poplar bark lipids enhance mouse immunity by inducing T cell proliferation and differentiation. The Journal of Veterinary Medical Science, 82(8), 1187-1196.

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Molecular Analysis of Cell Signaling Pathways

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Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/Streptomycin (Cat #15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) and G418 sulfate were obtained from Invitrogen (Carlsbad, CA). Rabbit antibody against β-actin (Cat #4970S) was from Cell Signaling (Beverly, MA). Rabbit antibody against NHE1 (Cat #ab67314) and rat antibody against CD8 (Cat #ab22378) were from Abcam Ltd. (Cambridge, MA). Rat anti-mouse CD31 (Cat #550274) was from BD Pharmingen (San Jose, CA). Rabbit anti-ionized calcium-binding adapter molecule 1 (iba1) was from Wako (Richmond, VA). PerCP/Cy5.5-CD45, PE-P2RY12, BV421-TGFβ, BV605-TNFα, APC/780-IL-1β, PE/Cy7-IL10, PerCP/Cy5.5-CD8a, APC/Cy7-CD4, Alexa Fluor 700-CD25, PE-FoxP3, BV605-PD-1, PE-Gr-1, APC-NK1.1, and Pacific Blue Granzyme B were obtained from Biolegend (San Diego, CA). eFluor 450-CD16/32, PE/Cy7-CD206, APC-IFNγ was purchased from eBioscience (San Diego, CA). APC-IL-6 were obtained from thermos fisher (Waltham, MA). BUV737-CD11b, Alexa Flour 700-CD86 were obtained from BD Biosciences (San Jose, CA). PE-Ym1were from Abcam Ltd. (Cambridge, MA). Rabbit anti-Laminin (Cat #L9393) and cariporide (HOE642) was purchased from Sigma Chemicals (St. Louis, MO). Anti-mouse PD-1 (RMP1-14) and IgG2a isotype control were from BioXcell (West Lebanon, NH).

Guan X., Luo L., Begum G., Kohanbash G., Song Q., Rao A., Amankulor N., Sun B., Sun D, & Jia W. (2018). Elevated Na/H exchanger 1 (SLC9A1) emerges as a marker for tumorigenesis and prognosis in gliomas. Journal of Experimental & Clinical Cancer Research : CR, 37, 255.

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Tumor-Infiltrating Lymphocyte Isolation and Characterization

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TILs isolation was done as previously described [21 ]. TDLNs were processed, suspended in RPMI-1640, then washed and filtered. Tumor cells or TILs were blocked with anti-mouse CD16/32 (FcR blocker; BioLegend, Cat#101302) for 10 min at room temperature, and then stained with Pacific blue-CD45 (BioLegend, Cat#103126), APC/Cy7-CD4 (BioLegend, Cat#100414), PerCP/Cy5.5-CD8a (BioLegend, Cat#100734), PE-CD11c (BioLegend, Cat#117308), FITC-F4/80 (BioLegend, Cat#123108), APC/Cy7-CD11b (BioLegend, Cat#101226), and PE/Cy7-Gr-1 (BioLegend, Cat#108416) at room temperature for 30 min. For intracellular staining cells were fixed and permeabilized according to the manufacturer’s instructions (eBioscience, Cat#00-5523-00) and stained with PE-FOXP3 (eBioscience, Cat#12-5773-82) and Alexa Fluor 647-IDO1 (BioLegend, Cat#654004). All samples were analyzed with LSRII flow cytometer and FlowJo software (version 10.0.7).

Li A., Barsoumian H.B., Schoenhals J.E., Cushman T.R., Caetano M.S., Wang X., Valdecanas D.R., Niknam S., Younes A.I., Li G., Woodward W.A., Cortez M.A, & Welsh J.W. (2018). Indoleamine 2,3-dioxygenase 1 inhibition targets anti-PD1-resistant lung tumors by blocking myeloid-derived suppressor cells. Cancer letters, 431, 54-63.

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Modulation of MDSCs and CD8+ T cells

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Isolated MDSCs were cultured in the indicated conditioned medium for 24h, followed by FACS analysis using MDSC markers PerCP/Cy5.5-CD11b (BioLegend #101228), APC-Ly6G (BioLegend # 127613), Arg1 (Cell Signaling #93668S) and iNOS (Cell Signaling #13120S). The purified 5×10⁵ CD8⁺ T cells were mixed with pre-washed Dynabeads Mouse T-Activator CD3/CD28 (Life Tech #11456D), and cultured in the indicated conditioned medium for 24h, followed by FACS analysis using CD8⁺ T cell markers PerCP/Cy5.5-CD8a (BioLegend #100733), FITC-IFN-γ (BD Biosci # 554411) and PE-Ki67 (BioLegend #151209).

Zhao D., Cai L., Lu X., Liang X., Li J., Chen P., Ittmann M., Shang X., Jiang S., Li H., Meng C., Flores I., Song J.H., Horner J.W., Lan Z., Wu C.J., Li J., Chang Q., Chen K.C., Wang G., Deng P., Spring D.J., Wang Y.A, & DePinho R.A. (2020). Chromatin Regulator, CHD1, Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer. Cancer discovery, 10(9), 1374-1387.

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