Rat tnf α quantikine elisa kit

Pluronic F-127 was provided by Funakoshi Co., Ltd. (Tokyo, Japan), and Tra powder and CA-TRA were kindly donated by Kissei Pharmaceutical Co., Ltd. (Nagano, Japan). A Rat TNF-α Quantikine ELISA Kit was purchased from Bio-techne (Seattle, WA, USA), and fluorescein was obtained from Alcon (Tokyo, Japan). Pluronic F68, Evans blue (EB), isoflurane, ethyl p-hydroxybenzoate, and D-mannitol (Man) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Benzalkonium chloride (BAC) was provided by Kanto Chemical Co., Inc. (Tokyo, Japan), and MC was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). HPβCD and Carbopol (Carbopol^® 934) were obtained from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and Serva (Heidelberg, Germany), respectively. Cell Count Reagent SF was purchased from Nacalai Tesque Inc. (Kyoto, Japan), and penicillin, Dulbecco’s Modified Eagle’s Medium/Ham’s F12, heat-inactivated fetal bovine serum (FBS), and streptomycin were provided by GIBCO (Tokyo, Japan). A Bio-Rad Protein Assay Kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals and organic solvents were of analytical grade.

Rats were euthanized by injecting a lethal dose of pentobarbital, and the conjunctiva were excised and homogenized in saline on ice. The homogenates were centrifuged at 20,400 g for 20 min at 4°C, and the supernatants were used for the measurement of TNF-α levels. TNF-α levels were measured using a Rat TNF-α Quantikine ELISA Kit (Bio-techne, Seattle, WA, United States) according to the manufacturer’s instructions. The TNF-α levels are expressed as pg/mg protein.