Il 1b

To quantify cytokine concentrations in cell free supernatants of lung tissue in vitro, ELISA (IL-18; MBL, Nagoya, Japan) and multiplex-assay (IL1b, IL6, IL8, IL10, IL12, IL17A, G-CSF, GM-CSF, TNF-α; Bio-Rad, München, Germany) were used after incubation of 0.3–0.4 g pieces of lung tissue at 37 °C and 5% CO₂ in 2 mL RPMI medium (RPMI 1640 + 10% FCS) for 24 h. Cytokine assays were done according to kit instructions and were performed on 11 of the 13 lung specimens due to tissue shortage after pathological assessment in the remaining cases.

Cytokine levels in the vaginal swab samples were quantified using Bio-Rad Bio-Plex Pro human cytokine 8-Plex assay kit (Bio-Rad Laboratories, Inc., USA) with a Luminex 3D system. In addition, a single-Plex assay was used for IL-1B (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (Sharma et al., 2009 (link)). Frozen vaginal swab samples were thawed, vortexed vigorously for 1 min and the swab squeezed on the wall of the tube to maximize elution. The swab samples were then centrifuged at 700 x g for 10 min. The Bio-Rad assay was performed using samples and serial dilutions of standards in duplicate, according to the manufacturer’s instructions. Cytokine levels were analyzed using LuminoXponent software. Chemokine and cytokine values for samples with levels below the lower limit of detection (LOD) were assigned the lower limit of detection for the specific cytokine. The lower limits of detection ranged from 0.01 to 1.1 pg/ml. The median proportion of samples below the LOD was 1.85% (ranging from 0% to 25.4%).