Bioluminescence assay kit cls 2

Caudal epididymal sperm were isolated in TBS, pH 7.4 as described. Sperm were incubated with or without energy substrates for 1 hour at 37°C. Triplicate 50-μl aliquots were diluted into 450 μl of boiling Tris-EDTA buffer (0.1 M Tris-HCl and 4 mM EDTA; pH 7.75) as described previously (34 (link)). The diluted suspensions were boiled for 5 min and then frozen on dry ice. The frozen samples were thawed and centrifuged at 15000 × g for 5 min at 4°C. The supernatant was then diluted (usually1:10) using the Tris-EDTA buffer and 100 μl of diluted sample was then utilized for quantifying ATP using the Bioluminescence Assay Kit CLS II (Roche Applied Science). Luminescence was measured in a Turner Biosystems 20/20 Luminometer. To determine the effect of external substrates on ATP levels, sperm were resuspended in pyruvate- and lactate-free TYH medium and incubated with 10 mM glucose or 25 mM lactate. After 2 hours of incubation triplicates aliquots were diluted 1:10 in boiling Tris-EDTA buffer (0.1 M Tris- HCl and 4 mM EDTA; pH 7.75 and used for ATP measurement as described above.