Apo one fluorescent substrate

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.

Activated caspase-3 and 7 activity in hind limb homogenate supernates was determined using the ApoONE fluorescent substrate (Promega, Madison, WI, USA). Briefly, individual, thawed muscle biopsies (80–100 mg biopsy) were mixed with 1.0 mL of 4 °C lysis buffer containing 50 mmol/L Na HEPES, pH 7.4, 100 mmol/L NaCl, 1 mmol/L ethylene diamine tetra-acetic acid, and 10% glycerol and homogenized at 4 °C for 30 s with an Omni tissue homogenizer. The muscle biopsy homogenates were centrifuged at 14,000× g (4 °C) in an Eppendorf microfuge for 20 min. The post-mitochondrial supernates were assayed for cytochrome C and caspase-3,7 activity, and the protein content was determined by the Bradford method using bovine serum albumin (BSA) as the standard. The activated caspase-3,7 assay was performed as follows: 50 μL of muscle supernate (≈150 μg of supernate protein) was mixed with 50 μL of the Apo-ONE substrate solution in the wells of a black, opaque, 96-well plate to initiate the 60 min reaction. The plates were shaken at 200 RPM at 37 °C for 60 min. The DEVD (Asp-Glu-Val-Asp peptide) caspase substrate peptide cleavage was measured using a Clariostar fluorescent plate reader (BMG Labtech, Cary, NC, USA) at an excitation wavelength of 499 nm and an emission wavelength of 521 nm. A caspase internal standard was included in each experiment.

Activated caspase 3 and 7 activity in kidney homogenate supernates was determined using the Apo-ONE fluorescent substrate (Promega, Madison, WI, USA) [16 (link)]. Briefly, thawed kidneys were mixed with an 8-fold excess of lysis buffer containing 50 mM Na HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 10% glycerol and homogenized at 4 °C for 30 s with an Omni tissue homogenizer. The kidney homogenate was centrifuged at 4 °C (16,000× g) in an Eppendorf microfuge for 20 min. The supernates were immediately assayed for caspase activity, and protein content by the Bradford method using bovine serum albumin 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 min reaction. Plates were shaken at 200 RPM at 37 °C for 60 min. The DEVD 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.