Cd14 positive selection kit

Mouse bone marrow macrophages were derived as previously described (16 (link)). Human monocytes were enriched using a CD14 positive-selection kit (Miltenyi Biotech, Auburn, CA). Culture media was replaced with antibiotic-free medium the night before the assay. Rapa groups were pre-treated with rapamycin at indicated concentrations for 2hr prior to infection. Chloroquine (Sigma) was used at 20μM as a positive control. Lm was stained with pH-insensitive e670 dye (eBioscience, San Diego, CA) to identify infected cells. Cells were infected (MOI=5-10) for 5 min, treated with gentamycin, then washed twice before staining with lysotracker (Molecular Probes, Eugene, OR). Infected cells were identified as e670+ and then the ratio of the geometric mean fluorescence intensity of acidic to neutral was calculated to determine relative lysosomal acidification.

Blood was collected in EDTA tubes from patients with loss-of-function IL-10R mutations and control subjects (either a healthy parent or an unrelated healthy donor) following protocol approval and in accordance with the Declaration of Helsinki. Blood samples were shipped at room temperature overnight to our lab at Boston Children’s Hospital and upon arrival PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare, Pittsburgh, PA) gradient, according to manufacturer’s instructions. Monocytes were sorted using CD14 positive selection kit (Miltenyi Biotec) and cultured in RPMI 1640 supplemented with 20% FCS and antibiotics. To generate M1 macrophages media was supplemented with 100 ng/mL GM-CSF for 8 days (Rey-Giraud et al., 2012 (link)), and for M2 macrophages media was supplemented with 50 ng/mL of M-CSF for 7 days and an additional day with 20 ng/mL of IL-4 (Hedl and Abraham, 2012 (link)).