Human il 8 elisa max standard set

MDA-MB-231 cells (5 × 10⁵) were plated in 60 mm dishes. After serum deprivation overnight, the cells were stimulated with LPA for 24 h. Each inhibitor was added for 30 min before LPA stimulation. Conditioned medium was harvested, and secretion of IL-8 was determined using the ELISA MAX Standard Set Human IL-8 (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. The absorbance at 570 nm was measured using an iMark Microplate Reader (Bio-Rad, Hercules, CA, USA). The amount of IL-8 was calculated from a standard curve and normalized to the total protein content.

Cytokine and chemokine profiling was done by using the Bio-Plex Pro™ 27-Plex Human Cytokine Panel (Bio-Rad, Hercules, CA, USA), the Bio-Plex Pro™ 40-Plex Human Chemokine Panel (Bio-Rad, Hercules, CA, USA) and the single-Plex TSLP (Bio-Rad, Hercules, CA, USA). For all plexes, all cytokines and chemokines were multiplexed on the same 96-well plate (1 plate for each plex). Cytokine and chemokine standards were serially diluted, and protein profiling from all challenges were done as per the manufacturer’s instructions (Bio-Rad), with 4 biological replicates. Quality controls (from the kit) were also included, only for the 40-plex, to ensure the validity of the results obtained. Protein concentrations were calculated by using the Bio-Plex ManagerTM software and expressed in pg/mL.
IL-8 was quantified by using ELISA Max™ Standard Set Human IL-8 (BioLegend, San Diego, CA, USA); manufacturer instructions were followed.

For assessing immunomodulation of intestinal cells, HT-29 cells were co-incubated with bacteria at a multiplicity of infection (MOI) of 40 or media, with recombinant TNF-α (Peprotech, London, UK) at a final concentration of 5 ng/mL as previously described (for more details see Supplementary Materials). Supernatants were recovered and stored at −80 °C until subsequent analysis. Interleukin-8 (IL-8) was later quantified in the supernatants using the Human IL-8 ELISA MAX Standard Set (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Each strain was tested 3 times from 3 independent cultures.
Immunomodulation of PBMCs was assessed in cells from five healthy donors (for more details, see Supplementary Materials). Bacteria at an MOI of 10 or control media were added for 24 h. Supernatants were recovered and stored at −80 °C until subsequent analysis. IL-10 and IL-12p70 were later quantified in the supernatant by ELISA (Mabtech, Cincinnati, OH, USA) according to the manufacturer’s instructions. Each strain was tested 3 times from 3 independent cultures.