Bms603 2

The serum levels of inflammatory markers, including IL-1β, IL-6 and TNF-α, were detected using commercial mouse TNF-α (cat. no. BMS607-3; Invitrogen; Thermo Fisher Scientific, Inc.), IL-1α (BMS611; Invitrogen; Thermo Fisher Scientific, Inc.) and IL-6 (BMS603-2, Invitrogen; Thermo Fisher Scientific, Inc.) ELISA kits. A total of 50 μl serum samples were added to the prepared microwells of the plate and diluted twice with the sample solvent. To quantify the relative protein levels, standard solutions of various concentrations were added to additional wells instead of samples. To the resulting mixtures, 50 μl diluted biotin conjugate was added and the mixture was incubated for 2 h at room temperature. Subsequently, 100 μl streptavidin-HRP was added and incubated for 1 h at room temperature. The microwells were washed multiple times and stained with tetramethylbenzidine. The intensity of staining was assessed using an EFOS-9305 spectrophotometer (Shvabe Photosystems, Moscow, Russian Federation, https://www.shvabe.com) at a wavelength of 620 nm. The concentration of substances was calculated on the basis of a calibration curve constructed from the data of a titration of standard solutions. Detection of inflammatory cytokines was performed three times.

Plasma cytokine concentrations were assessed using Mesoscale Discovery mouse V-Plex kits (K15048 and K15245) following the manufacturer’s protocol. Limits of detection can be found in the Supplementary Appendix (http://links.lww.com/CCX/B131). In the initial survival study, 6-hour plasma IL-6 levels were assessed by nonlethal blood sampling (10 mcL) and enzyme-linked immunosorbent assay (Invitrogen, Waltham, MA, BMS603-2) per the manufacturer’s protocol.