Multiplex kits

Serum levels of 94 analytes was analyzed using Bio-Plex (Bio-Rad, Hercules, CA, USA) multiplex magnetic bead-based antibody detection kits following manufacturer’s instructions. Multiplex kits (Bio-Rad, Hercules, CA, USA), Bio Plex Pro Human Cytokine 21-plex, Bio Plex Human Cytokine 27-plex, Bio Plex Human Cytokine 37-plex, and Bio-Plex Human MMP Panel panels were used in the study. Data collected was analyzed using with MasterPlex CT control software and MasterPlex QT analysis software (MiraiBio, Alameda, CA, USA).

Blood was collected from anesthetized (1–5% isoflurane, Abbott Laboratories, Chicago, IL, USA) mice via the inferior vena cava using a 23 G needle. After collection, blood was transferred to Capiject serum separator tubes (Terumo Medical Corp., Elkton, MD, USA), allowed to clot for 30 min at room temperature, and centrifuged at 400 g for 10 min. The serum was collected and stored at –70°C until used. Luminex 200 (Luminex Corp., Austin, TX, USA) was used to detect cytokines, chemokines, and growth factors in mouse serum samples supernatants as described earlier using multiplex kits (Bio-Rad Inc., Hercules, CA, USA)^{(15 (link))}. Mice administered amifostine were treated with 50, 100 or 150 mg/kg dose. Serum samples were collected 4, 8, 24 and 48 h post amifostine administration. Cytokines were evaluated in mice treated with 25 and 50 mg/kg of GT3 and blood samples were collected 24 h post-GT3 injection.