Ammonium chloride potassium lysis buffer

Human monocytes were obtained from buffy coats of healthy volunteers from the Belgian Red Cross-Flanders. The mononuclear cells were isolated from the buffy coats using Ficoll (Sigma-Aldrich). CD14 + monocytes were isolated using MACS separation following manufacturer's instructions (Miltenyi Biotec). The monocytes were seeded in RPMI with 10% inactivated human serum, 1% nonessential amino acids, 1% sodium pyruvate and 1% glutamine. After two days in culture, human IFN-α was added to the cells during three days to obtain hSn-positive monocyte-derived macrophages (MDMs). BALB/c mice were sacrificed and their primary bone marrow cells were collected in RPMI medium. Red blood cells were removed using an ammonium-chloride-potassium lysis buffer (VWR and Janssen Chimica). Macrophages were seeded on coverslips in a 24-well plate in RPMI with 10% inactive fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% glutamine and with addition of L929 supernatant. L929 supernatant was retrieved from L929 cells, kindly provided by dr. C. Uyttenhove (Ludwig Institute for Cancer Research, Brussels, Belgium). After three days in culture, mouse interferon-alpha (IFN-α) was added to the cells, to increase the number of Sn-positive macrophages.

CHO-K1 cells were kindly provided by Dr. J. D. Esko (University of California, San Diego, CA, USA) and were transfected or transduced to obtain stable cell lines expressing hSn (hSn + -CHO) and mSn (mSn + -CHO) [37] . These cells were subcultured in RPMI with 10% heat inactivated fetal bovine serum (iFBS). Primary mouse bone marrow macrophages were collected in RPMI medium. After removing red blood cells with an ammonium-chloridepotassium lysis buffer (VWR and Janssen Chimica), macrophages were seeded in RPMI with 10% iFBS, 1% non-essential amino acids, 1% sodium pyruvate, 1% glutamine and with addition of L929 supernatant and 500 U/ml mouse IFN-α (R&D) in the medium. L929 supernatant was collected from cells kindly provided by Dr. C. Uyttenhove (Ludwig Institute for Cancer Research, Brussels, Belgium). Human monocytes were obtained from blood of healthy volunteers from the Belgian Red Cross-Flanders. The monocellular cells were isolated from the blood using Ficoll (Sigma-Aldrich). CD14 + monocytes were isolated using MACS separation following manufacturer's instructions (Miltenyi Biotec). The cells were seeded in RPMI with 10% heat inactivated human serum, 1% non-essential amino acids, 1% sodium pyruvate and 1% glutamine. After two days in culture, 50 ng/ml human IFN-α was added for three days to obtain hSn-positive macrophages.