Tc 20 cell counter system

RAW cell line was acquired from American Type Culture Collection (ATCC). These cells were unfrozen and were cultured at 1 × 10⁶ cells in a TC-25 flask (Costar, ON, Canada) with RPMI 1640 medium supplemented with 10% FBS (GIBCO BRL, Grand Island, NY, USA), 100 unit/mL penicillin, 10 μg/mL streptomycin and 250 ng/mL amphotericin B (Sigma-Aldrich; Saint Louis, MO, USA).
After a 2-day incubation period (37 °C, 5% CO₂), cells were collected, and viability was determined by trypan blue exclusion using a TC-20 cell counter system (Bio-Rad, Hercules, CA, USA). The cells in culture were routinely 90 to 95% live cells. The cells were used in passages 10–15, according to previous reports [19 (link)].

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors at the blood bank of the National Institute of Respiratory Diseases in Mexico City. PBMCs were collected and counted to determine their viability using the TC20 cell counter system (Bio-Rad, California, USA). Monocytes were enriched by employing positive selection with magnetic microbeads coated with CD14 antibody (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity was confirmed by flow cytometry, and the purified cells were found to be 90%–95% of the intended cell type.