Fitc labeled antibodies

Human blood samples from healthy anonymized donors giving informed consent were collected into EDTA by a certified phlebotomist according to the guidelines and approval of California State University Long Beach Institutional Review Board. Primary human monocytes were isolated using the Dynabeads Untouched Human Monocyte Kit from Invitrogen (Carlsbad, CA) according to the manufacturer’s protocol. Cell purity was determined using the Scepter cell analyzer (EMD Millipore, Darmstadt, Germany). Isolated monocytes were used in phagocytosis assays (purity >93%) or cultured for 7-13 days in RPMI 1640, 10% FCS, 2-mM L-glutamine and 1% penicillin/streptomycin containing 25 ng/ml rhM-CSF (Peprotech, Rocky Hill, NJ) to stimulate differentiation in human monocyte derived macrophages (HMDM). Expression of macrophage markers CD11b and F4/80 were assessed by flow cytometry using FITC-labeled antibodies (eBioscience, San Diego, CA) to characterize and validate macrophage differentiation and were >94% population for each experiment.

Raw264.7 cells (ATCC), a murine macrophage cell line, were cultured in DMEM supplemented with 10 % Fetal Calf Serum (FCS), 100 U/ml Penicillin and 100 μg/ml Streptomycin (Invitrogen), as described [27 (link)]. C57bl6 control mice are commercially available (Jackson Laboratory). For obtaining bone marrow-derived macrophages (BMDM), murine femurs were dissected and flushed out with DMEM-2 % FCS. Non-adherent cells were cultured in DMEM supplemented with 10 % FCS, 15 % L929 conditioned medium, Pen/strep in 5 % CO₂ as described in detail in [32 (link)]. Expression of macrophage markers CD11b and F4/80 were assessed by flow cytometry using FITC-labeled antibodies (eBioscience, San Diego, CA, USA) to characterize and validate macrophage differentiation and only cells >90 % positive for those markers were used for each experiment.