Human tnf α elisa kit

The cell culture supernatant of each group was collected and centrifuged at 1000 × g for 20 min, and then the supernatant was transferred into new tubes, detected immediately, or stored in an aliquot at −80°C for later use. The amounts of IL‐10 and TNF‐α in the supernatant were detected by using the Human IL‐10 ELISA Kit (mlbio) and Human TNF‐α ELISA Kit (mlbio), respectively, according to the manufacturer's instructions. Briefly, 50 μL of standard or sample was added to the wells, and 100 μL of enzyme conjugate was added to the standard and sample wells, covered with an adhesive strip, and incubated for 60 min at 37°C, after which the plate was washed four times with wash solution. Fifty microliters of substrate A and 50 μL of substrate B were added to each well. The samples were gently mixed and incubated for 15 min at 37°C in a dark environment. Fifty microliters of stop solution was added to stop the reaction, and then the plate was read at 450 nm by using a microtiter plate reader within 15 min.