Mouse ifn γ elisa kit

According to the manufacturer’s guidelines (Invitrogen), the levels of TNF-α, IFN-γ, and CXCL10 in the culture supernatants were evaluated using a Mouse TNF-α ELISA Kit, a Mouse IFN-γ ELISA Kit, and a Mouse CXCL10 ELISA Kit, respectively.

Lung leukocytes (5 × 10⁵) were stimulated in 96-well flat-bottom plates with CD3/CD28 anti-mouse antibodies using PBS as a control and cultured at 37 °C with 5% CO₂. Four days following stimulation, the culture supernatants were carefully removed after pelleting the cells and stored at −80 °C until the determination of IFN-γ concentration using the mouse IFN-γ ELISA Kit (Invitrogen, MA, USA) according to the manufacturer’s guidelines.

Blood samples were collected through tail vein sampling or cardiac puncture prior to perfusion. Each cardiac puncture sample was divided for serum collection by centrifugation, plasma collection was carried out using Lithium heparin collection tubes (365985, BD Biosciences, Franklin Lakes, NJ, USA). Owing to the small sample sizes, tail vein samples were reserved solely for serum isolation. All samples were spun at 2000 g, at 4°C for 15 min, after which the supernatant was collected, and aliquots taken and frozen at −80C.
Serum IFN-γ levels were quantified using a commercially available mouse IFN-γ ELISA kit (50-173-19, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Serum sample aliquots were sent to the BIDMC Small Animal Imaging Facility (LSAIF) for potentiometric quantification of BUN, ALT, and AST (Vitros 350 Chemistry System, Ortho Clinical Diagnostics). Serum creatinine quantification was performed using isotope dilution liquid chromatography - tandem mass spectrometry (LC-MS/MS) at the UAB/UCSD O'Brien Center Core C. For western blotting, plasma was diluted 1:10 in 1× protease and phosphatase inhibitors in H₂0, and SDS-PAGE was carried out as described above.