Imark microplate absorbance reader spectrophotometer

Specific ELISA kits were employed to determine the tear levels of various proteins, including IFN-γ (cat. no. 900-k27), IL-1β (cat. no. 900-k47.) and IL-8 (cat. no. 900-k18; all obtained from Peprotech, Inc., Rocky Hill, NJ, USA). Briefly, 96-well plates were coated with the primary antibody at a concentration of 0.5 µg/ml (1:200) prior to being incubated overnight at room temperature. Following aspiration and washing (4 times) of wells 300 µl blocking buffer (1% bovine serum albumin in PBS) was then added and the plate was incubated for 1 h. Following aspiration and washing, the standard solution or 10 µl of tear sample was added to wells in duplicate and incubated for 2 h. Then the plate was washed 4 times and the detection secondary antibody (biotinylated) at 0.5 µg/ml was added and incubated during 2 h at room temperature. After that, avidin peroxidase (1:2,000) from the same ELISA kit was added and incubated for 30 min at room temperature, followed by the addition of 100 µl 2,2-azine-bis (3-ethylbenzothiazoline-6-sulfonic acid). The absorbance was subsequently measured at 415 nm with the wavelength correction set at 650 nm in an iMark™ microplate absorbance reader spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Finally, the cytokine levels were calculated by linear regression analysis.