Gentamicin

In order to obtain human monocytes, PBMCs were cultured in a 75 cm² cell culture flask for 2 h at 37 °C in a 5% CO₂ environment at a concentration of 3–4 × 10⁶ cells/mL in X-VIVO 15 media (Lonza, MD, USA) supplemented with 1% inactivated autologous serum, gentamicin (50 μg/mL, B/Braun Medical, Melsungen, Germany), fungizone (2.5 μg/mL, Life Technologies, California, USA), and Zidovudine (1μM, Genéricos Españoles Laboratorios, Madrid, Spain). Monocytes were confirmed to be in a monolayer after incubation for 2 h, and the nonadherent cells (MNC) were removed by three washes with phosphate-buffered saline (PBSx1) (Figure S1). Subsequently, monocytes were cultured for 6 days with 1000 U/mL each of recombinant human IL-4 (ProSpec, Rehovot, Israel) and recombinant human GM-CSF (ProSpec) on days 0 and 2. After 6 days, immature dendritic cells (iDCs) were obtained by adding cold PBSx1 to the 75 cm² cell culture flask to the harvest them by firmly tapping the flasks. This dislodged the loosely adherent dendritic cell (DC) clusters. MDDCs were harvested into a 50 mL conical tube and counted manually for viability using the trypan blue exclusion dye. Lymphocytes were collected from MNC fractions and frozen as PBMCs for use in mixed lymphocyte response assays across different DC preparations.

After isolation, PBMCs were immediately cultured in a T75 flask for 2 h at 37 °C in a humidified air 5% CO₂ atmosphere at a concentration of 3–4 × 10⁶ cells/mL in X-VIVO 15 media (Lonza, Basel, Switzerland), supplemented with 1% human AB serum (Sigma-Aldrich), 50 μg/mL gentamicin (B/Braun Medical, Melsungen, Germany) and 2.5 μg/mL amphotericin B (fungizone; Bristol-MyersSquibb, Rueil-Malmaison, France). After 2 h of incubation, monocytes were observed as adherent cells and nonadherent cells were removed by three washes with PBS 1X. Subsequently, 1000 U/mL each of human recombinant IL-4 and GM-CSF (both from ProSpec, Rehovot, Israel) were added to monocytes (days 0 and 2) and after 5 days the immature MDDCs were obtained and washed with cold PBS 1X.