Mouse il 10 elisa max deluxe

ELISA kits (Mouse IL-6 ELISA MAX™ Deluxe, Mouse IL-10 ELISA MAX™ Deluxe, and Mouse TNF-α ELISA MAX™ Deluxe, Biolegend, Mouse MCP-1/CCL2 Uncoated ELISA kit, Mouse TGF-beta 1 ELISA kit, Invitrogen) were used in this study. According to the manufacturer’s protocol, capture antibody solution was firstly added into all wells of a 96-well plate provided in the kit. The plate was subsequently sealed and incubated at 4°C overnight. After washing and blocking, 100 μL of each standard and cell culture supernatant sample was added into the wells and incubated for 2 hrs at room temperature. The wells were then incubated with detection antibody for 1 hr, followed by washing and incubation with HRP-labeled Avidin for 30 minutes. TMB mixture (1:1) was then added for 20 minutes in the dark to visualize chemiluminescence. Then, 100 μL of 2 M sulfuric acid solution was added in each well to stop the reaction. The reading of ELISA results was carried out using a plate reader (SpectraMax i3, Molecular Devices) by recording the absorbance at 450 nm within 15 mins after adding the stop solution.

The validation of cytokine measurement was carried out by measuring PBS solutions spiked with different concentrations of cytokines (mouse IL-4, IL-6, IL-10, TNF-α, ebioscience, USA) using both LSPR microarrays and the gold-standard ELISA. ELISA kits (mouse IL-4 ELISA MAX™ Deluxe, Mouse IL-6 ELISA MAX™ Deluxe, Mouse IL-10 ELISA MAX™ Deluxe, and Mouse TNF-α ELISA MAX™ Deluxe, Biolegend) were used in this study according to manufacturer’s protocols. Briefly, cytokine solutions or cell culture supernatants were collected as samples. Capture antibody solution was added into all wells of a 96-well plate provided in the kit, the plate was subsequently sealed and incubated at 4°C overnight. After washing and blocking, 100 μL of each standards and samples were added into the wells and were incubated for 2 hours at room temperature. The wells were then incubated with detection antibody for 1 hour, followed by washing and incubation with HRP-labeled Avidin for 30 minutes. TMB mixture (1:1) was then applied to visualize chemiluminescence for 20 minutes in the dark. Then, 100 μL of 2N sulfuric acid solution was added in each well to stop the reaction. The reading of ELISA results was carried out using a plate reader (SpectraMax i3, Molecular Devices) by reading the absorbance at 450 nm within 15 mins after adding the stop solution.