Histopaque 1077 separation gradient

Human tissue was weighed, minced with a razor blade and digested with 30 μg/mL Liberase TM, 50 μg/mL DNase I (all from Roche, Burgess Hill, UK) in RPMI for 30 minutes at 37 °C (rocker incubator). After digestion, tissue was transferred into gentleMACS™ C-tubes and dissociated using programme ‘Spleen-0’ and ‘Lung-02’ on gentleMACS™ Dissociator (Miltenyi Biotech, Bisley, UK). Homogenized tissue was filtered through 100-μm and 40-μm Falcon™ cell strainers (Fisher Scientific, Loughborough, UK). After one wash in running buffer (PBS, 2 mM EDTA, 0.5% (w/v) BSA), the cell suspension was further refined by using 44% Percoll® (GE Healthcare Life Sciences, Little Chalfont, UK) separation gradient and Dead cell removal kit (Miltenyi Biotech, Bisley, UK).
Human splenic tissue was weighed and then homogenized using 70-μm Falcon™ cell strainer (Fisher Scientific, Loughborough, UK) in a running buffer. Repeated red cell lysis was then performed as described in murine experiments. Human peripheral mononuclear cells (PBMCs) were isolated from 9 mL of full blood (with EDTA anticoagulant) using Histopaque®-1077 separation gradient (Sigma-Aldrich, Gillingham, UK). After cell counting, splenic suspension or PBMCs were directly stained for FACS/cytometry without any further purification steps.

Monocytes were obtained from the peripheral blood of healthy donors from the Bahia State Blood Center (HEMOBA). Blood was diluted in saline (1:1) and processed using the HISTOPAQUE^® 1077 separation gradient (Sigma Aldrich, St Louis, MO) for 30 minutes at 360xg at room temperature. After the centrifugation, a ring consisting of mononuclear cells was formed, collected and washed 3 times with saline at 200xg, 4°C. For differentiation into DCs, mononuclear cells were passed through magnetic separation columns for isolation of CD14⁺ cells (monocytes). These CD14⁺ cells were cultured for 7 days at a concentration of 1x10⁶/mL in complete RPMI medium (Gibco Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 2mM L-glutamine, 100U/mL of penicillin and 100μX/mL streptomycin (complete medium) in the presence of IL-4 (100UI/mL) and GM-CSF (50ng/mL; both from PeproTech, Rocky Hill, NJ, USA) in 24- wells plates. Every 3 days of culture, 500 μL of medium containing growth factors were removed and replaced with the same amount of fresh medium.