Ab100697

We measured the serum levels of IL-6 in the blood samples using commercial mouse kits (ab213749 and ab100697; Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. We added 50 μL of samples to each of the wells in 96-well antibody-coated plates and then incubated these for 2 h at room temperature. We then loaded 50 μL of the detector antibody solution into each well and incubated the plates for an additional 1 h at room temperature. Next, we added 50 μL of HRP–streptavidin solution (ab210901; Abcam) to each well and once again incubated the plates for 1 h at room temperature. Finally, we added 100 μL of tetramethylbenzidine substrate to each well and incubated the plates for another 10 min in the dark at room temperature. We stopped the reaction by adding 100 μL of stop solution. We read the absorbance at a wavelength of 450 nm and expressed the results in pg/mL.

The blood samples were used to measure serum levels of IL-6 and IL-10 using mice commercial kits (ab213749 and ab100697; Abcam, Cambridge, MA, USA) following the manufacturer instructions. Samples of 50 μL were added to each well of the 96-well antibody-coated plates and then incubated for 2 h at room temperature. The detector antibody solution (50 μL) was loaded into each well and the plates were incubated for 1 h at room temperature. Next, 50 μL of the HRP-Streptavidin solution (ab210901, Abcam) was added, and the plates were incubated for another 1 h. Then, 100 μL of TMB substrate was added to each well and the plates were incubated for 10 min in the dark. The reaction was stopped with the addition of 100 μL stop solution. A wavelength 450 nm was used to read the absorbance and the results are expressed in pg/mL.

The concentrations of TNF-α and IL-10 in the supernatants of the culture medium were determined by ELISA kits (ab100747, ab100697, Abcam, USA) following the manufacturer's instructions. The culture medium was collected at day 3 and centrifuged at 1,500 rpm for 20 min at 4°C to remove cell debris, and the supernatant was used for protein quantification.