Hcd14 apc

CD34+ HSPCs were seeded at a density of 2 × 10⁵ cells/ml in non-treated 6-well plates in differentiation medium (SFEM II supplemented with SCF (200 ng/ml), Il-3 (10 ng/ml), IL-6 (10 ng/ml), FLT3-L (50 ng/ml), M-CSF (10 ng/ml) and penicillin/streptomycin (10 U/ml)). After 48 h, non-adherent cells were removed and reseeded in a new non-treated 6-well plate at 2 × 10⁵ cells/ml in differentiation medium. Adherent cells were maintained in the same dish in maintenance medium (RPMI supplemented with FBS (10% v/v), M-CSF (10 ng/ml) and penicillin/streptomycin (10 U/ml)). After 2 weeks, adherent macrophages were harvested by incubation with 10 mM EDTA in PBS. For phenotypic analysis, 1 × 10⁵ cells per condition were harvested and resuspended in 100 μl staining buffer comprises PBS supplemented with 2% FBS and 0.4% EDTA. Non-specific antibody binding was blocked (5% v/v TruStain FcX, BioLegend, #422302) and cells were stained with 2 μl of each fluorophore-conjugated monoclonal antibody (30 min, 4 °C, dark). Antibodies used were hCD34-APC (BioLegend, #343509), hCD14-BV510 (BioLegend, #301842) or hCD14-APC (Invitrogen, #17-0149-41), and hCD11b-PE (BioLegend, #101208). Propidium Iodide (1 μg/ml) was used to detect dead cells and cells were analyzed on a BD FACSAria flow cytometer.