Apc anti human cd14

Ficoll Isopaque was obtained from the LUMC Pharmacy (Leiden, Netherlands). Fluorochrome-conjugated antibodies were purchased from several suppliers. V500 anti-mouse CD3, FITC anti–mouse CD8, FITC anti–human CD4, Pacific Blue anti–human CD8, and APC anti–human CD14 were purchased from BD Biosciences. BV605 anti–mouse CD8 was purchased from BioLegend. Fluorochrome-conjugated streptavidin and 7-AAD were purchased from Invitrogen. DAPI was purchased from Sigma. Conventional HLA–A*02:01 PE-labeled tetramers were produced by K.L.M.C. Franken, M.G.D. Kester, and L. Hageman (LUMC, Leiden, Netherlands) as described previously (Altman et al., 1996 (link)). Human IL-2 was purchased from Chiron. Human serum albumin was purchased from Sanquin Reagents.

Surface staining (phenotyping) was performed using fresh peripheral blood collected in the lavender tube with EDTA, BD Vacutainer. A 150-μL sample of fresh blood was used for each test tube (Th1, Th17, Treg,). Blood was incubated with a specific antibody mix for each population, for 30 min in the dark.
Th1: FITC anti-human CCR5 (BD), PE anti-human CXCR3 (Biolegend), APC anti-human CD4 (BD);
Th17: FITC anti-human CCR6 (Biolegend), PE anti-human CD161 (BD), APC anti-human CD14 (BD);
Treg: FITC anti-human CD4 (BD Pharmigen), PE anti-human CD25 (BD Pharmigen), APC anti-human CD127 (Biolegend).
Subsequently, FACS lysing (BD) was added to remove red blood cells (15 min, in darkness at room temperature). Finally, cells were washed with cold FACS Flow (BD) then resuspended in 1 mL of FACS Flow (BD). Ten thousand gated CD4+ cells were acquired by Cell-Quest four colors Facs Calibur cytometer (BD). The analysis of the results was carried out using the FlowJo software v.8.4.