Anti cd14 fitc antibody

Buffy coats from healthy donors were collected in the Blood Center of the Centro Médico Nacional 20 de Noviembre or in the Blood Center of the Instituto Nacional de Ciencias Médicas y Nutrición Mexico, under the approval of the Institutional Ethics Committee. Human monocyte-derived macrophages (hMDMs) were purified from peripheral blood mononuclear cells (PBMCs) from freshly collected blood samples. PBMCs were isolated by density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich, USA) according to the manufacturer’s recommendations. PBMCs were washed with PBS 1X and cultured in RPMI-1640 medium supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY) for 2 h at 37°C in a humidified atmosphere with 5% CO₂ to allow monocytes to adhere to the plastic plate. Non-adherent cells were eliminated by washing, and adherent cells, enriched for monocytes, were cultured for 7 days for macrophage differentiation in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded at either at 6×10⁴, 5×10⁵ or 1.2×10⁶ cells per well in 96, 12 or 6-well tissue culture plates, respectively. Differentiation was confirmed by determining the CD14 expression by flow cytometry using an anti-CD14-FITC antibody (BD Biosciences).

Human peripheral blood mononuclear cells were isolated from buffy coats obtained from the local blood bank (permit nr DP086) using Ficoll-Paque Plus density gradient centrifugation (GE Healthcare). Briefly, the samples were layered over 15 ml of Ficoll and centrifuged brake-free for 25 min at 800 g at room temperature. The interface was removed and washed twice with PBS containing 2% FBS and 1 mM EDTA. The cells were resuspended in PBS containing 2% FBS and 1 mM EDTA. The monocytes were enriched using the EasySep™ Human CD14 Positive Selection Kit (Stemcell Technologies) according to the manufacturer's instructions. The purity of isolated monocytes was tested by flow cytometry immediately after isolation by staining with anti-CD14-FITC antibody (BD Biosciences) and was >97% in all experiments.