Rosettesep human monocyte cell enrichment cocktail

RosetteSep Human monocyte Cell Enrichment Cocktail (Stemcell; cat. #15,028) was used according to the manufacturer’s protocol to isolate monocytes by negative selection from the blood of anonymous healthy donors. Donors signed a clinical consent form for the donation procedure, which includes language that the donor center can direct use of all materials and by-products for clinical or research use. All procedure involving peripheral blood mononuclear cells (PBMCs) from anonymous donors were reviewed and approved in DF/HCC IRB-exempted protocol 93–011. Monocytes were seeded in 96-well flat bottom plates (Costar; Cat. #3596) in X-VIVO 15 medium (Lonza; Cat. #04418Q) at 0.6 × 10⁶/well and different cytokines were added.

U-937, THP-1, and HeLa human cell lines (ATCC), and human and murine primary cells, were grown in RPMI or DMEM (Sigma) supplemented with fetal calf serum (FCS, 10%), 4 mM l-glutamine, 1 mM sodium pyruvate, penicillin (100 U/mL), and streptomycin (0.1 mg/mL) (Biological Industries, Israel) at 37°C and 5% CO₂. U-937 and THP-1 monocytes were differentiated to macrophages with 5 ng/mL PMA (Cayman Chemical, Ann Arbor, MI, USA) for 3 days. Primary human CD4⁺ T cells and monocytes were purified from buffy coats by the RosetteSep Human CD4⁺ T or the RosetteSep human monocyte cell enrichment cocktail according to the manufacturer’s instructions (StemCell Technologies, USA). Primary monocytes were differentiated to macrophages (confirmed by morphology and adhesion) with 10 ng/mL GM-CSF (Peprotec, Rehovot, Israel) for 6 days.