Opteia set human il 6

PBMC, 4x105 cells per well, were cultured in 48 wells plate in presence of RPMI 1640 (Gibco, 61870- 010) completed with 5% of heat inactivated AB human serum (Sigma, H4522). IL-6 secretion was assessed in PBMC culture supernatant 2, 15 and 24 h after recombinant Spike exposure, by ELISA using BD Opt EIA Set Human IL-6 (BD, 555 220) according to supplier’s recommendations. To analyze a possible contribution of endotoxin in the stimulation of IL-6 response, cells were treated during 24 h with a combination of 10 ng/mL LPS-EK (Invitrogen, Tlrl-eklps), 12.5 μg/mL Polymyxin B (inhibitor of endotoxin, Invitrogen, Tlrl-pmb), 2 μg/mL recombinant non-stabilized trimer Spike SARS-CoV-2 (ACROBiosystems, USA; SPN-C52H8) or its buffer in quantities equivalent to 2 μg/mL. IL-6 was measured (triplicates) on culture cell supernatants using the IL-6 ELISA assay kit BD Opt. EIA human IL-6 ELISA set (BD, 555 220) and GloMax plate reader (Promega, GM3000).

This experiment was performed as described in the previous section, but C. acnes fresh inoculum was added instead of TNF-α. C. acnes was grown until the stationary phase (5 days incubation under anaerobiosis in BHI culture medium). Then bacterial cells were harvested and diluted in FSB-free DMEM medium, adjusted to OD 1.2 at 550 nm. Different dilutions of this inoculum were added to HaCaT cells and incubated overnight. Quantification of IL-6 in cell culture supernatants was assayed by using ELISA Human kit (BD OptEIA^® Set Human IL-6, BD Biosciences, Franklin Lakes, NJ, USA) following manufacturer instructions [44 (link)].