Anti cd62l fitc

After 24, 48, 72, and 120 h-cultures, cells were washed twice in sorter buffer (PBS containing 1% FCS [Gibco BRL, USA] and 0.1% sodium azide [Sigma]) and blocked for 10 min with PBS containing FC-receptor bloking solution (BioLegend Inc., San Diego, CA, USA), then stained for 30 min in the dark with fluorochrome-conjugated monoclonal antibodies (mAbs) against the relevant cell surface antigens to characterize main leukocyte populations (anti-CD3 FITC, PE, and PerCP; anti-CD4 PerCP and APC; anti-CD8 PerCP; anti-CD14 FITC; anti-CD83 APC; anti-CD80 PerCP; and anti-CD86 PE) and cell activation status (anti-CD69 PE and Cy and anti-TLR2 APC) and to identify T cell phenotypes as naïve, memory, or effector (anti-CD45RO APC and anti-CD62L FITC). All mAbs were obtained from Becton Dickinson (BD Biosciences). After incubation at 4°C, cells were washed twice for 10 min, first with sorter buffer and then with PBS containing 0.1% sodium azide. Cells were then fixed with 1% paraformaldehyde (Sigma) and stored at 4°C in the dark. Cells were collected within 48 h on the FACSAria flow cytometer (BD Biosciences). Isotype controls were used to define the negative cell region, and 50,000 events were acquired for each sample. The data were analyzed using FlowJo version 8.2 software (Treestar, Ashland, OR, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation on a Ficoll–Paque PLUS gradient (GE Healthcare, Uppsala, Sweden). Neutrophils were further isolated after dextran sedimentation and hypotonic lysis as previously described (20 (link)).
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.

Antibodies against CD3 and CD28-coupled magnetic beads (anti-CD3/28), penicillin, and streptomycin were from Invitrogen (Grand Island, NY, USA). Anti-CD62L-FITC, anti-CD45RA-TC, anti-CD14-Pacific Blue, anti-PD-1-APC, anti-HLA-DR-Alexa Fluor® 700, CD69-PE, and CD16-FITC were from Biolegend (San Diego, CA, USA). Anti-human CCL4-PerCP and its mouse IgG2B-PerCP isotype control were from R&D systems (Minneapolis, MN, USA). HindIII,BamHI,XhoI,NotI, and BstEII were from New England BioLabs (Ipswich, MA, USA). Puromycin and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).