Cd4 allophycocyanin apc

The procedure used in this study has been published previously (14 (link),26 (link)). Briefly, the following anti-mouse monoclonal antibodies were purchased from BD Biosciences: Ly6G-peridinin chlorophyll protein (PerCP)-Cy5.5 (1A8; 560602), Ly6C-fluorescein isothiocyanate (FITC) (AL-21; 553104), CD4-allophycocyanin (APC) (RM4-5; 553051), CD25-FITC (7D4; 553071), and CD19-APC (1D3; 550992). The following antibodies were purchased from BioLegend: CD45-FITC (30F-11;103108), CD45-PerCP-Cy5.5 (30F-11;103132), CD45-brilliant violet (BV510) (30F-11;103138), CD11b-APC/Fire750 (M1/70;101262), CD244-Alexa Fluor 647 (2B4;133509), F4/80-phycoerythrin (PE) (BM8;123110), PD-L1-BV421 (10F.9G2;124315), CD8a-APC/Fire 750 (53-6.7;100766), CD8a-PE (53-6.7; 100708), CD127-BV510 (A7R34;135033), CD1d-PE (1B1;123509), and CD5-BV421 (53-7.3;100618). The isolated immune cells were stained in a 96-round-bottom-well plate for 20 min at 4°C and washed with PBS containing 1% bovine serum albumin. Sorting of Ly6G⁺CD244⁺ and Ly6G⁺CD244⁻ cells was conducted on a FACSAria instrument (BD Biosciences). Flow cytometric data were obtained by a FACS Verse instrument (BD Biosciences) and analyzed by FlowJo software (TreeStar, Inc.). CD45-gated cells were analyzed in this study.

CD4⁺CD45RO⁻CD62L⁺ central memory T-cell isolation was performed on a Miltenyi AutoMACS Pro. Thirty million peripheral blood mononuclear cells (PBMCs) were labeled with CD4⁺ Central Memory T Cell Biotin Cocktail (Miltenyi Biotec, Auburn, CA) followed by antibiotin microbead. The negative selection antibody cocktail contained antibodies against CD8, CD14, CD15, CD16, CD19, CD25, CD36, CD45RA, CD56, CD123, TCRγ/Δ, and CD235a (glycophorin A). Cells recovered from the negative selection fraction were then labeled with anti-CD62L phycoerythrin (PE) followed by anti-PE MicroBead positive selection of CD62L⁺ cells. Purity was assessed by flow cytometry using antibodies reactive to CD3 (peridinin chlorophyll protein [PerCP]; BD Biosciences, San Jose, CA), CD4 (allophycocyanin [APC]; BD Biosciences), CD45RO (fluorescein isothiocyanate [FITC]; BD Biosciences), and CD62L (PE; BD Biosciences). Purified T cells were stored at −80°C.

The cell concentration of lymphocytes was adjusted to 1x10⁶ cells/ml using PBS (Beijing Solarbio Science & Technology Co., Ltd.) and 100 µl suspension was added to a flow tube. After incubating with CD3-FITC (BD Pharmingen; BD Biosciences) 20 µl and CD4-allophycocyanin (APC; BD Pharmingen; BD Biosciences) 20 µl for 15 min in the dark, 400 µl PBS was added. The % of CD3⁺/CD4⁺ T lymphocytes was isolated using a flow cytometer (BD Pharmingen; BD Biosciences) and then analyzed using FlowJo version 7.6 software (FlowJo LLC).