Burkitt's lymphoma Raji cells (ATCC® CCL-86™) and SK-BR-3 breast tumor cell lines (ATCC® HTB-30™) were obtained from American Type Culture Collection. Raji cells were cultured in complete RPMI with 10% fetal bovine serum (FBS) and SK-BR-3 cells were cultured in complete DMEM with 10% FBS. The collections of CHO cells expressing FLAG-tagged hFcγRs were described previously (14 (link), 40 (link), 45 (link)).
Human PBMCs and neutrophils were purified from anonymous healthy volunteers using Histopaque density gradient centrifugation (Sigma-Aldrich). Neutrophils were activated with 50 ng/mL hIFNγ for 24 h. NK cells were isolated by negative immunodensity isolation using the RosetteSep Human NK Cell Enrichment Cocktail (StemCell Technologies). Human GM-CSF differentiated macrophage cells were differentiated from CD14+ monocytes with 50 ng/mL GM-CSF (BioLegend®) for 7 days. Ectodomains of hFcγRI, hFcγRIIa, hFcγRIIb, and hFcγRIIIa, were produced in Expi293 cells (Invitrogen). All primers were synthesized by Integrated DNA Technologies.
Human PBMCs and neutrophils were purified from anonymous healthy volunteers using Histopaque density gradient centrifugation (Sigma-Aldrich). Neutrophils were activated with 50 ng/mL hIFNγ for 24 h. NK cells were isolated by negative immunodensity isolation using the RosetteSep Human NK Cell Enrichment Cocktail (StemCell Technologies). Human GM-CSF differentiated macrophage cells were differentiated from CD14+ monocytes with 50 ng/mL GM-CSF (BioLegend®) for 7 days. Ectodomains of hFcγRI, hFcγRIIa, hFcγRIIb, and hFcγRIIIa, were produced in Expi293 cells (Invitrogen). All primers were synthesized by Integrated DNA Technologies.
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