Mouse ccl 20 mip 3 alpha quantikine elisa kit

Chemokine (CCL-20/MIP-3α) levels in mixed glial cell culture supernatants were measured using a Mouse CCL-20/MIP-3 alpha Quantikine ELISA Kit (MCC200, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, 100 µL of the samples was added to each well and incubated for 2 h. After washing, 200 µL of human MIP-3α conjugate was added to each well and incubated for 2 h. The wells were washed, and 200 µL of substrate solution was added to each well and incubated. After addition of 100 µL of the stop reaction solution, the absorbances of the solutions in the wells were measured using a microplate reader.

The concentrations of leptin and insulin in serum, CCL-20 in the supernatent of cultured BMDM and albumin in the faeces were measured by enzyme-linked immunosorbent assays, with mouse standards, according to manufacturer’s guidelines.: Mouse Leptin ELISA (90030, Crystal Chem), Mouse Insulin ELISA (90080, Crystal Chem), Mouse CCL-20/MIP-3 alpha Quantikine ELISA Kit (MCC200, R&D Systems) and Mouse Albumin ELISA Quantification set (E90-134,Bethyl).

Snap-frozen mouse eyes were thawed in ice-cold lysis buffer, and retina was isolated under dissection microscope. The retinal tissue was homogenized and protein was extracted. Total protein concentration was measured using Bradford assay. Enzyme-linked immunosorbent assay (ELISA) was performed for detection of CCL20 in the tissue using Mouse CCL20/MIP-3 alpha Quantikine ELISA Kit (Cat # MCC200, R & D Systems, Minneapolis, MN) following the manufacturer’s instruction. Briefly, 50 μL assay diluent and 50 μL standard or samples were added to wells of a microplate pre-coated with monoclonal anti-mouse CCL20 antibody and incubated for 2 h at room temperature allowing thorough mixing. The wells were washed and incubated with monoclonal mouse CCL20 conjugated with horseradish peroxidase for 2 h at room temperature. The wells were treated with substrate reagent and read at 450 nm with correction at 540 nm using a Synergy H4 microplate reader.