Pan monocytes isolation kit

A549 (human lung carcinoma), HT1080 (human fibrosarcoma), and HeLa (human epithelium) cells were obtained from the American Type Culture Collection (ATCC; Cat# CCL-1885, Cat# CC-121, and Cat# CCL-2), and were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 29.2 mg/ml L-glutamine (Life Sciences), and 5 μg/ml plasmocin (Invivo Gen, San Diego, CA), in a humidified incubator with 5% CO₂ at 37 °C. Human THP-1 and U937 monocytes and human Jurkat T cells were obtained from ATCC (Cat# TIB-202, CRL-1593.2 and Cat#TIB-152) and maintained in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% FBS and 1% penicillin/ streptomycin/ L-glutamine in a humidified incubator with 5% CO₂ at 37 °C. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donor using the Pan Monocytes Isolation kit from Miltenyi Biotec (130-096-537). Monocytes were differentiated into macrophages by culturing in DMEM supplemented with 10% human serum for 7 days. The A549 cell lines that stably express the restriction factors TRIM5α_rh or TRIMCyp were previously generated (Selyutina et al., 2020). HeLa cell line stably expressing CPSF6 fused at C-terminal to the enhanced green fluorescent protein (eGFP) was generated by retroviral transduction.

Citrate blood was centrifuged for 30 min at 400×g. After the isolation of plasma, the buffy coat was resuspended in RPMI-1640 culture medium. The cell suspension was pipetted on Ficoll and was centrifuged for 40 min at 760×g. The resulting buffy coat was mixed with RPMI-1640 and centrifuged for 10 min at 760×g. This washing step was repeated 3 times. The resulting cells were sorted using the Miltenyi pan monocytes isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), containing a cocktail of biotin-conjugated monoclonal antibodies against antigens not expressed on human monocytes (negative sorting) as described in the manufacturer´s protocol. Briefly, 10 million cells were incubated at 4 °C for 5 min with FcR blocking reagent (human IgG) and the cocktail of biotin conjugated antibodies. Magnetic microbeads conjugated to monoclonal anti-biotin antibodies were added to the solution, incubated for 10 min at 4 °C and subjected to a subsequent magnetic cell separation through a magnetic field. Finally, the monocyte fraction was collected and used for cell culture and further analyses.