Ac lehd afc

The activities of caspase-3/7, -8 and -9 were measured in total cell lysates of Jurkat cells (1×10⁶/ml) treated with ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml), in the absence or presence of catalase (1000 U/ml) as required and for the time period indicated, using an appropriate fluorescent substrate.^{44 (link)} Ac-DEVD-AFC (Bachem) was used as a substrate for caspase-3/7, Ac-IETD-AFC (Bachem) for caspase-8 and Ac-LEHD-AFC (Bachem) for caspase-9. Fluorescence was monitored continuously for 30 min using a fluorescence microplate reader (Tecan Safire2) at an excitation wavelength of 405 nm and emission wavelength of 535 nm. Results are presented as change in fluorescence as a function of time.

Apoptosis in tumors was analyzed by detection of caspase activity, which was measured by cleavage of specific fluorogenic substrates: Ac-DEVD-7-amino-4-trifluoromethyl coumarine (afc) (Bachem, Heidelberg, Germany) for caspase-3, Ac-LETD-afc (Alexis, Grünberg, Germany) for caspase-8 and Ac-LEHD-afc (Bachem) for caspase-9. After homogenization of tumors, corresponding fluorogenic substrate was added. Fluorescence intensity was measured in a Shimadzu RF-5301PC fluorometer. Caspase activity was calculated from the slope as fluorescence units/mg protein/minute of reaction time and converted to picomoles of substrate cleaved/mg protein/minute based on a standard curve for afc.