Comparison of the DTNB Sample Clauses

Comparison of the DTNB and OPA assays Prior to commencing the experiments in chapters 3 and 4, the DTNB and OPA assays were both used to measure the concentration of thiols extracted from BAEC with modified thiol concentration and the results were compared. BAEC were cultured as described above and seeded into 24 well plates. When confluent the medium was replaced with medium containing BSO (10, 25 and 100 µM) for 24 hrs or left untreated. After each treatment period, the medium was aspirated and xxxxx were washed twice with ice-cold phosphate buffered saline (PBS; 10 mM, pH 7.4). ice-cold trichloroacetate (TCA) (6.5%; 100µL) was added to each well for 10 mins on ice, aspirated and stored at -70oC. 100 µL NaOH (1M) was added to each well for 2 hrs before aspirating and storing at - 20oC. 10 µL of each TCA extract was pipetted into the well of a clear and a black opaque microtitre plate. In addition to TCA extracts, a series of GSH (Sigma- Xxxxxxx, UK) solutions of known concentration were pipetted (10 µL) into each microtitre plate to construct a standard concentration curve. The DTNB and OPA assays were then carried out on the clear and black plates respectively.