Quantifying Kidney Caspase Activity

Activated caspase 3 and 7 activity in kidney homogenate supernates was determined using the Apo-ONE fluorescent substrate (Promega, Madison, WI).^{24 (link)} Briefly, thawed kidneys were mixed with an 8-fold excess of lysis buffer containing 50 mmol/L Na HEPES, pH 7.4, 100 mmol/L NaCl, 1 mmol/L ethylene diamine tetra-acetic acid, 10 mmol/L dithiothreitol, 10% glycerol and homogenized at 4°C for 30 seconds with an Omni tissue homogenizer. The kidney homogenate was centrifuged at 4°C (16 000g) in an Eppendorf microfuge for 20 minutes. The supernates were immediately assayed for caspase activity, and protein content by the Bradford method using BSA as the standard. The activated caspase assay was performed as follows: 5 µL supernate (≈40 μg of supernate protein) was diluted to a total volume of 50 µL with the above lysis buffer, was mixed with 50 μL of the undiluted Apo-ONE substrate in the well of a black, opaque, 96 well plate to initiate the 60-minute reaction. Plates were shaken at 200 RPM at 37°C for 60 minutes. The DEVD (Asp-Glu-Val-Asp peptide) caspase substrate peptide cleavage was measured using a BMG Clariostar fluorescent plate reader at an excitation wavelength of 499 nm and an emission wavelength of 521 nm. A caspase standard was included in each experiment.