Lympholyte poly cell separation media

Mouse peritoneal macrophages from C57BL/6 mice were collected as previously described [25 (link), 43 (link)]. Cells were cultured in RPMI-1640 supplemented with antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, Gibco, California, USA) and 10% fetal bovine serum. Human PBMCs were isolated according to a previous method [31 (link)]. Briefly, venous blood from healthy volunteers was collected into heparin-containing Vacutainer tubes (BD Biosciences, New Jersey, USA), diluted with an equal volume of PBS (pH 7.4) and separated by density gradient centrifugation using Lympholyte-poly cell separation media (Cedarlane, Ontario, Canada). Mononuclear cell layers were collected, washed and cultured in RPMI 1640 with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (Invitrogen, California, USA). Vero E6 cells were gifted by Dr Chunsheng Dong and cultured in DMEM supplemented with antibiotics and 10% FBS. All cells were cultured in a humidified incubator under 5% CO₂ at 37 °C.

Buffy coats were obtained from healthy blood donors at Transfusion Medicine of the National Cancer Institute of Naples, after informed written consent. Human peripheral blood mononuclear cells were harvested by density gradient centrifugation of 50 mL buffy coats mixed with an equal volume of PBS using Lympholyte-poly Cell Separation Media (Cedarlane Laboratories, Milan, Italy). Serum and peripheral blood mononuclear cells (PBMC) were individually collected. PBMCs were washed twice with PBS, counted using the trypan blue dye exclusion method, and monocytes isolated by positive selection of CD14⁺ cells with the Monocyte Isolation Kit II (Miltenyi Biotec Bologna, Italy). The obtained monocytes (88% pure by visual and cytofluorimetric analysis) were transferred to tissue culture plates in RPMI-1640 medium, supplemented with 10% autologous human serum, penicillin (100 U/mL) and streptomycin (100 μg/mL). To obtain murine monocytes, blood samples (about 500 μL/mouse) from the retro-orbital venous plexus of mice anesthetized with 1% isoflurane, were collected using a heparinized capillary tube. PBMCs purified from whole blood with the OptiPrep^TM gradient solution (Sigma-Aldrich), were transferred to tissue culture plates in RPMI-640 medium supplemented with 10% heat-inactivated FBS for three days to isolate adherent monocytes/macrophages.

Buffy coats were obtained from healthy blood donors at Transfusion Medicine of the National Cancer Institute of Naples, after informed written consent. Peripheral blood mononuclear cells (PBMC)s, and serum were individually collected. PBMCs were harvested by density gradient centrifugation as previously described [29 (link)], using the Lympholyte-poly Cell Separation Media (Cedarlane Laboratories, #CL5015, Cedarlane, Burlington, ON, Canada) according to the manufacturer’s instructions. Monocytes were isolated from PBMCs by positive selection of CD14+ cells using the Monocyte Isolation Kit II purchased by Miltenyi Biotec, #130-091-153 (79% pure by visual and cytofluorimetric analysis) and transferred to tissue culture plates in RPMI-1640 medium (Cytiva HyClone™ #SH30096.01, Cytiva, Marlborough, MA, USA), supplemented with 10% autologous human serum, penicillin (100 U/mL), and streptomycin (100 μg/mL).