2.6. Monocyte Isolation

The separation of cells occurred from 10 mL of the total blood in tubes with 3 mL of Histopack 1077 and 3 mL of Histopack 1119 after 30 min of centrifugation at 400× g. The layer corresponding to leukocytes was transferred to sterile conical tubes and diluted in PBS. The tubes were centrifuged again at a speed of 400× g at 4 °C for 10 min. Subsequently, a 1 h incubation was performed on monocytes adhered to the plaque. The supernatant was composed of 2 mL of culture medium RPMI-1640 (enriched with 1 mL of serum from the volunteer and 650 µL of penicillin) and 500 mL cell suspension leukocytes in the wells. The total number of cells used in the culture assay was 1 × 10⁶ in each well. After cell separation, monocytes were stimulated with 0.2 mL of LPS 5μg/mL and incubated for a period of 24 h, in a greenhouse, at 37 °C, at a concentration of 5.0% CO₂. All supernatants were aliquoted in Eppendorf tubes of 2000 µL and frozen in the freezer at −80 °C until analysis. Brand reagents Sigma-Aldrich (Merck group), San Luis, MI, USA.