Pe cy7 conjugated anti cd3

For primary NK cells, single hepatic lymphocytes from 6- to 8-week-old C57BL/6 mice were labeled with PE-Cy7-conjugated anti-CD3, PE-conjugated anti-CD4, APC-conjugated anti-NK1.1 and APC-Cy7-conjugated anti-CD19 (BD PharMingen, United States). NK cells were sorted as cells that express NK1.1⁺CD3^- using a FACS Aria I flow cytometer (BD Biosciences, United States) and the purity was higher than >95%. After purification, NK cells were cultured in a 96 round bottom wells (Costar, Corning Incorporated, Corning, NY, United States) in complete RPMI 1640 medium supplemented with 10% (v/v) fetal serum (Gibco, United States), 200 mg/ml glutamine, 50 μg/ml gentamycin, 100 units/ml penicillin, 100 μg/ml streptomycin (Sangon Biotech, Shanghai, China) and interleukin-2 (IL-2, 10 U/ml, BD PharMingen, United States).

Immediately after CS exposure and lung removal, lung tissues were digested with collagenase and DNase (Sigma, St. Louis, MO, USA) to prepare single-cell suspensions by passing the dissociated tissue through a 70-μm cell strainer (BD Falcon). The suspensions of cells were stained in PBS containing 2% FBS with the following antibodies: FITC-conjugated anti-CD4, PE-Cy7-conjugated anti-CD3, APC-Cy7-conjugated anti-CD45, PE-conjugated anti-CD69, and APC-conjugated anti-CD8 antibodies (BD Bioscience). The cells were gated on FSC (forward scatter) and SSC (side scatter). The cells were then gated on SSC versus CD45 followed by SSC versus CD3 in order to gate on all T cells. The cells were further gated to obtain the percentage of CD4+ T and CD8+ T cells. Stained cells were examined on a FACS Canto flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The percentages of activated CD4+ T cells and activated CD8+ T cells were determined based on double positive cells (CD69+ CD4+ or CD69+ CD8+ cells) among total CD4+ or CD8+ T cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. The following monoclonal antibodies (mAbs) and reagents were used in this study: PE-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD4, PE-conjugated anti-CD38, APC-conjugated anti-HLA-DR, APC-Cy7-conjugated anti-HLA-DR, FITC-conjugated anti-CD45RA, PerCP-Cy5.5-conjugated anti-CCR7 (BD Biosciences, USA); Violet-conjugated anti-CD38, FITC-conjugated anti-CD38, PE-Cy7 conjugated anti-CD25, APC conjugated anti-CD69, APC conjugated anti-CD127, Amcyan-conjugated anti-CD45RA (BioLegend, San Diego, CA, USA). For the expression of all markers, flow cytometric gating was defined using fluorescence minus one (FMO) controls. CD4⁺ T cell subsets were identified in terms of CD45RA and CCR7 expression. CD38 and HLA-DR were measured on gated CD4⁺ T cell subsets: naive CD4⁺ T cells (Tn, CD3⁺CD4⁺CD45RA⁺CCR7⁺), central memory CD4⁺ T cells (Tcm, CD3⁺CD4⁺CD45RA⁻CCR7⁺), and effector memory CD4⁺ T cells (Tem, CD3⁺CD4⁺CD45RA⁻CCR7⁻). The expression of CD25, CD69, and CD127 were measured on gated CD38⁺ Tcm, HLA-DR⁻ Tcm, and CD4⁺HLA-DR⁺ cells. Data were collected using a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo software (TreeStar, USA).