Specific elisa kit

Blood was withdrawn by tail vein puncture (1.0 mL into heparin-containing 1.5 mL tubes, on day 0) or after decapitation (6–8 mL into heparin-containing 15 mL tubes, on day 21 or 42), centrifuged at 3500× g, 4 °C for 10 min. Plasma was separated by aspiration using a narrow-necked pipette, inserted into heparin-containing tubes, and kept at −80 °C until further assessment. Li, creatinine, and urea levels were detected in the Biochemistry lab of Soroka University Medical Center (SUMC), Beer-Sheva, Israel. Li levels were measured using an Ion-Selective Electrode (ISE) electrolyte analyzer (Cobas Integra 400, Roche Diagnostics, Rotkreuz, Switzerland); creatinine and urea were measured using an Olympus Beckman Coulter AU5800 apparatus (Brea, CA, USA). Cystatin C levels were determined by a specific ELISA kit (Boster Bio, Pleasanton, CA, USA).

Culture supernatants collected 24 h and 1 week post stimulation were assayed using Multiplex kits (Thermo Fisher Scientific, USA). The specific kit used for the 24-h culture supernatant contained the following analytes: IL-10, IL-6, IL-8, IL-29, CCL-2, TNF-α, IFN-α, CXCL-10, and CCL-19. The kit for the 7-day supernatants contained these analytes: TNF-α, IFN-α, IL-6, IFN-γ, IL-17A, IL-1β, IL-22, and granzyme B. The procedure followed was according to the protocol of the manufacturer. Briefly, the supernatant was mixed with premixed beads (30 cytokines) overnight, and after incubation with detection antibodies and streptavidin-PE for 1 h each, the plate was run on Magpix to identify specific cytokines. IL-18 was detected using specific ELISA kit (Boster Bio, CA, USA).

The concentrations of IL‐1β and IL‐6in different time points were measured using specific ELISA kits from Boster Biological Technology according to the manufacturers’ protocols.