CCR2 FACS Antibody Using Primary Monocytes

One day prior to the experiment, isolate monocytes using Ficoll gradient centrifugation. Afterwards, use magnetic bead separation to extract primary monocytes (negative selection). Incubate 1 Million cells in 2 ml RPMI (10% FCS, 2% glutamine) with or without 5 ng/ml PMA for 24 hours. Count cells, incubate 500,000 cells with 2 µl of the antibody in FACS buffer (1% BSA, 0.01% Sodium Azide) for 30 min on ice in the dark. Wash three times with 3 ml of FACS, buffer each time. During centrifugation, keep the cells at 4°C.