Apc conjugated anti cd14

Fresh heparinized whole blood from healthy donors was utilized for neutrophil, CD14, and CD2 purifications. Whole blood was lysed (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA in distilled water) for 10 min at RT and washed twice for 10 min at 300 × g. CD66b⁺ neutrophils were isolated by positive selection using the CD66abce Microbead kit (Miltenyi Biotec) according to manufacturer’s protocol. Purity was assessed by flow cytometry using BV421-conjugated anti-CD66b and APC-conjugated anti-CD14 (BD Biosciences). The purity of the enriched population was >98%. CD14⁺ and CD2⁺ cells were positively isolated from the PBMC fraction obtained by Ficoll-Hypaque Plus gradient separation (GE Healthcare, France), using CD14 and CD2 Microbeads (Miltenyi Biotec), according to manufacturer’s protocol. Briefly, CD14⁺-labeled cells were first enriched on positive selection columns and the depleted fraction containing total lymphocytes was washed and stained with CD2 Microbeads and isolated on positive selection columns. Purity of CD14 and CD2 enriched fractions was assessed by flow cytometry using APC-conjugated anti-CD14 (BD Biosciences) and efluor450-conjugated anti-CD3 (Ebioscience, France) and was >98%. Viability of the samples following each purification procedure was assessed by Trypan blue staining and was >97% for all experiments.

Ficoll-isolated monocytes (500,000 cells) from fresh PB and BM samples (after the 1–2 days preculture) were stained with 1.25 µl allophycocyanin (APC)-conjugated anti-CD14 (BD Biosciences, Franklin Lakes, NJ; Cat. No. 561383), 5 µl phycoerythrin (PE)-conjugated anti-CD16 (BD Biosciences; Cat. No. 555407), and 1.25 µl fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibodies (Invitrogen; Cat. No. 11-0036) for 20 min at RT. Ficoll-isolated CB monocytes (500,000 cells) were stained with above-mentioned amounts of APC-conjugated anti-CD14, PE-conjugated anti-CD16 and 2.5 µl FITC-conjugated anti-CD45 antibodies (BD Biosciences; Cat. No. 555482) for 20 min at RT. Samples were run on the BD Accuri 6C Plus (BD Biosciences) flow cytometer and analyzed with FlowJo v10.7.1 software (FlowJo LLC.).