Apotarget caspase colorimetric protease assay sample kit

ApoTarget caspase colorimetric protease assay sample kit (KHZ1001, Invitrogen Corp., Camarillo, CA, USA) was used to determine the activity of caspase-2, 3, 6, 8 and 9. Treated and untreated cells (5 × 10⁵ cells/well in 6-well plate) were collected and transferred into sterile test tube and lysed using the cell lysis buffer supplied. Samples (50 μL) of the lysate were aliquoted into wells of a standard black 96-well microplate, to which 50 μL of reaction buffer containing 10 mM dithiothreitol were then added to the sample wells. The selective substrates used; VDVAD-pNA, DEVD-pNA, VEID-pNA, IETD-pNA and LEHD-pNA substrates for caspase-2, caspase-3, caspase-6, caspase-8 and caspase-9, respectively were added to the appropriate wells (5 μL of 4 mM; final concentration 200 μM) and the plate was then incubated at 37°C for 4 h. The absorbance at 405 nm was measured in a microplate reader (Thermo Labsystem Multiscan Spectrum, Thermolabsystem, Chantilly, VA, USA). To determine the change in caspase activity, the absorbance of treated samples compared with untreated control group.

Apo Target caspase colorimetric protease assay sample kit (Catalog No. KHZ1001, Invitrogen Corp., Camarillo, CA, USA) was used to determine the activity of caspase-2, -3, -6, -8 and -9. Cells (5 × 10⁵ cells/well in 6-well plate) (treated and untreated) were collected and transferred into sterile test tube and lysed using the cell lysis buffer supplied. Samples (50 μL) of the lysate were aliquoted into wells of a standard black 96 well-microplate, to which 50 μL of reaction buffer containing 10 mM DTT were then added to the sample wells. Substrates selective for each of the caspase forms (5 μL of 4 mM; final concentration 200 μM) were added to the appropriate wells and the plate was then incubated at 37°C for 2 h. Absorbance at 405 nm was then read in a microplate reader.
The absorbance from treated samples was compared with untreated control to allow determination of the change in caspase activity. The selective substrates VDVAD-pNA, DEVD-pNA, VEID-pNA, IETD-pNA, and LEHDpNA were used for caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9, respectively.