Escherichia coli (DH5α, Invitrogen) was cultured overnight at 37°C in SOC broth. Bacteria were washed once in PBS and fixed in 2% paraformaldehyde for 20 min, then washed extensively before counting by Flow Cytometry (FACSVerse, BD Biosciences) and added to the THP1 (a human monocytic cell line derived from an acute monocytic leukemia patient) in a bacteria/cell ratio of 100:1 for 24 h. PBMCs were cultured for 1 h in round-bottom 96-well plates in the presence of E. coli-activated THP1 (1:2) or PMA (250 ng/μl) plus ionomycin (1 μg/μl) as positive control. Brefeldin A (10 μg/ml) was added and cells were incubated for further 4 h. Cells were harvested and stained as previously described. For the intracellular quantification of cytokine production, the following antibodies were used: IL-17 PE (Miltenyi Biotech), IFNγ PE and TNFα FITC (BD Biosciences).
The assay setup for both PMA/iono and bacterial stimulation was optimized on 2 HIV– and 1 HIV+ subjects. During each round of experiments, PBMCs from the same batch of the same HIV– subject were included. This allowed us to verify that the culture conditions were reliable and that the stimuli were working as expected.
The assay setup for both PMA/iono and bacterial stimulation was optimized on 2 HIV– and 1 HIV+ subjects. During each round of experiments, PBMCs from the same batch of the same HIV– subject were included. This allowed us to verify that the culture conditions were reliable and that the stimuli were working as expected.