Mouse il 6 duoset elisa kit

Mouse TNFα DuoSet ELISA kit (R&D systems, catalog number DY410, lot number P189768), mouse IL-6 DuoSet ELISA kit (R&D systems, catalog number DY406, lot number P234212), mouse IFNγ DuoSet ELISA kits (R&D systems, catalog number DY485-05, lot number P234214) and mouse IL-1β (Invitrogen, catalog number 88-7013-88 and lot number 183204000) were used to perform the assays. Glass-bottom 96-well black plate (P96-1.5H-N, Cellvis, Mountain View, USA) was first coated with capture antibodies as per manufacturer’s instructions (100 μl/well) and incubated overnight at 4°C. The plate was washed three times with 1x PBST (1x PBS with 0.05% Tween-20) and then blocked with 200 μl of reagent diluent (1x PBS in 1% BSA, 0.2μm filtered). After blocking the plates were washed three times with PBST, and serial dilutions of standard protein was added to different wells in duplicates and incubated for 2 hours at room temperature. The plates were washed three times with PBST and then incubated for 2 hours with 100 μl of biotinylated detection antibody as per the manufacturer’s instructions. The plates were washed three times with PBST and streptavidin Cy5-plasmonic-fluor (extinction 0.5) for 30 minutes at room temperature in dark. Finally, the plates were washed three times in PBST and imaged using Nikon TsR2 epifluorescence microscope.

IL-6 ELISAs were performed on supernatant from mouse lung homogenates using the Mouse IL-6 Duoset ELISA kit from R&D Systems (catalog # DY406) according to manufacturer’s instructions.