Fitc conjugated anti human cd14 antibody

Human PBMCs were re-suspended for cell sorting. We selected CD14+ monocytes/macrophages by using CD14 MicroBeads and Isolation Kit as previously reported [18 (link)]. Unmarked lymphocytes were also collected after magnetic cell sorting. Subsequently, monocytes/macrophages were treated with a FITC-conjugated anti-human CD14 antibody (Biolegend, CA, USA). The purity of monocytes/macrophages was confirmed by flow cytometry (BD FACSCanton-II, BD Bioscience, Franklin Lakes, NJ, USA) following the manufacturer’s protocol. Data were analyzed with FlowJo 7.6 software.

THP-1 monocytes (2 x 10⁶ cell/well in 6-well plates) were differentiated with VitD3 or PMA, as described in section 2.7. To prepare cell for flow cytometry analysis, both suspension and attached cells were collected from the plate at 0, 48, 72 and 96 h into centrifuge tubes and spun down. Then 100 μL of cell suspensions in microcentrifuge tubes containing 2x10⁵ cells were incubated with 2 μL of FITC-conjugated anti-human CD14 Antibody (BioLegend Way, California, USA) for 15 min at 4°C, followed by washing with 900 μL of PBS. Then, the tubes were centrifuged at 700 x g for 5 min to remove the supernatant, and, subsequently, the cells were stained with 1 μL of Propidium iodide (PI) (Cat. P3566, Life Technologies Corporation, USA) in 400 μL of PBS. For flow cytometry, a minimum of 10,000 events were acquired on an Attune™ NxT Acoustic Focusing cytometer (Thermofisher Scientific, USA), using Attune™ NxT v3.1.2 Software for data analysis. CD14 and PI were analyzed using the BL-1 and YL-2 channels, respectively. Quadrant regions indicated the percentage of cells for each sub-population.