Mouse tnf α elisa kit

A subset of TNF-α^Flox/Flox, LyzMcre^−/− (TNF-α^F/F) and TNF-α^Flox/Flox, LyzMcre± (TNF-α^F/F,LyzMCre) mice were administered 100ng of lipopolysaccharide (LPS) (Sigma Aldrich, St. Louis, MO) intraperitoneally and were subsequently sacrificed 1hour after LPS administration. Whole blood was collected via the inferior vena cava and plasma was assessed for circulating TNF-α utilizing a mouse TNF-α ELISA kit (BioLegend, San Diego, CA).

The release of cytokines into the cell culture supernatants and BALF were detected by enzyme-linked immunosorbent assay (ELISA). The IL-1β, TNF-α, and IL-18 levels were detected using a mouse IL-1β ELISA Kit (R&D Systems, Inc., Minneapolis, MN, USA), rat IL-1β ELISA Kit (Boster, Wuhan, People’s Republic of China), rat IL-18 ELISA Kit (Boster), and mouse TNF-α ELISA Kit (BioLegend, San Diego, CA, USA), following the manufacturer’s instructions.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and LPS (Escherichia coli serotype 055:B5) were purchased from Sigma–Aldrich, Saint Louis, MO, USA. Mouse TNF-α ELISA Kit, Mouse NO ELISA Kit and Mouse PGE2 ELISA Kit were obtained from Biolegend, San Diego, CA, USA. Protein COX-2, iNOS, p-ERK, ERK, p-NF-κB P65, NF-κB P65, p-IκBa and IκBa were purchased from CST, Danvers, MA, USA. Cell line RAW264.7 was provided by Cell Resource Center (IBMS, CAMS/PUMC, Beijing, China). BCA protein quantification kit was purchased from Beyotime company (Tianjin, China). PVDF membrane was provided by Roche company (Basel, Switzerland). Mouse RAW264.7 cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 pg/mL streptomycin. The cells were incubated at 37 °C under a 5% CO₂ and 90% relative humidity (RH) atmosphere.