Fitc conjugated anti cd8

Phenotypic maturation of DCs was analyzed by flow cytometry, cells were resuspended in PBS containing 2% FBS and stained with APC-conjugated anti-CD11c antibody, PC7-conjugated anti-CD40, PC5.5-conjugated anti-MHCII, FITC-conjugated anti-CD83 and PE-conjugated anti-CD86 anti-body (Biolegend, CA, USA). Non-treated DCs were also stained with isotype IgG as negative control. To collect DCs and CD3 co-cultured cells, splenocytes from the same mouse strain were obtained, and incubated with anti-CD3 beads followed by magnetic sorting (MACS). DC at 2 × 10⁴ cells/well from the BM of C57BL/6J were seeded in 96-well round-bottom plates, pulsed with Lewis cell lysates (5 μg/ml) and incubated with YYWY (50 or 100 μg/ml), LPS or un-treated at 5μg/ml was added and incubated with cells for 24 h at 37°C. CD3 cells per well and co-cultured with DC (2 × 10⁴/well) in 96-well round-bottom plate for 96 h. Then, the surface markers were stained with FITC-conjugated anti-CD8 (Biolegend, CA, USA), before lysis of the cell membrane with BD Cytofix/Cytoperm™ ContentsFixation, then the surface markers were stained with FITC-conjugated anti-CD8 (Biolegend, CA, USA), before lysis of the cell membrane with BD.

Fluorescently labeled antibodies or isotype-matched controls, including FITC-conjugated anti-CD8, APC-conjugated CD3, PE-conjugated anti-IL-17A, and PE-conjugated anti-IFN-γ, were purchased from BioLegend Company (San Diego, CA, USA). Briefly, fresh heparinized peripheral blood was incubated for 5 hours at 37°C in a humidified 5% CO₂ incubator by stimulation with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma) and ionomycin (1 μM, Sigma) in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). GolgiPlug (BD Biosciences) was subsequently added during the last 3 hours of incubation. Collected cells were washed three times with PBS and then surface-stained with anti-CD8 and anti-CD3 antibodies at 4°C for 30 minutes. Then, the cells were fixed and permeabilized using a FIX&PERM Kit (MultiSciences Biotech Co., Ltd.) according to the manufacturer's instructions, followed by staining with PE-conjugated anti-IL-17A or PE-conjugated IFN-γ. Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Peripheral blood samples were collected in morning fasting status. The fluorochrome-conjugated monoclonal antibodies used in this study were as follows: FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, APC-conjugated anti-CD25, APC-conjugated anti-CD28, BV421-conjugated anti-Foxp3, PE-conjugated anti-CD39, BV421-conjugated anti-TCRγδ, PE-conjugated anti-Vδ2, APC-conjugated anti-IL-17A, PE-Cy7-conjugated anti-IFN-γ (Biolegend, San Jose, CA, USA), PE-conjugated anti-PD-1 and PE-conjugated anti-PD-L1(BD Bioscience, San Jose, CA, USA), FITC-conjugated anti-Vδ1 (Abcam, USA), and relevant isotype controls. For extracellular staining, appropriate mAbs of surfacemarkers were added to samples in the dark at room temperature for 20 minutes. For intracellular staining, peripheral blood mononuclear cells (PBMCs) were stimulated with PMA (500 ng/mL), ionomycin (1 μg/mL), and BFA (2 μg/mL) at 37°C for 5 hours. For intranuclear staining, PBMCs were incubated with fixation/permeabilization buffer for 30 minutes at 4°C. Anti-Foxp3 antibody was added to the cell pellet for 30 minutes in the dark at 4°C. Labeled cells were then measured by a flow cytometer (BD Arial II; BD Bioscience, San Jose, CA, USA), and the data were analyzed via FlowJo 6.0 software (Tree Star).