After the reaction mixtures were incubated at 378C for 1 h, 7 amino 4 methyl coumarin separated from Ac DEVD MCA was calculated using a Docetaxel 114977-28-5 2350 fluorescence plate reader having an excitation wavelength at 360 nm and an wavelength at 460 nm, and expressed as change in fluorescence units FU.. Low specific proteolytic activity was determined in the presence of the CPP32 inhibitor Ac DEVD CHO, 1 mM. and specific CPP32 like proteolytic activity was obtained by subtraction of non specific activity from total activity. For diagnosis of ICE like activity, the same method was employed using Ac YVAD MCA and Ac YVAD CHO as an inhibitor and a substrate, respectively. Reduction of low KCl induced CPP32 activation by the drugs was expressed as 1ywFU low KCl plus drugs. yFU intact cells. xrwFU low KCl. yFU whole cells. x4 100 %.. Cellular ATP levels were determined according to the procedure accompanying the Sigma bioluminescent somatic cell assay kit. In quick, following the indicated treatment, cells were lysed with 0. 2 ml of ATP releasing barrier. ATP levels were measured using a microplate luminometer ML3000, Dynatech, Alexandria, VA.. ATP levels around 6 mM gave a standard curve and samples were tested in the linear range. Restoration of internal ATP standard within the products was 88. Cellular differentiation 7 2. 50-pound. All experiments were performed in triplicate wells and repeated many times with cultures from different platings. Data are shown as the mean S. N. Of-the averages of the individual tests. In some instances data are presented as the mean S. N. for representative experiment as described in figure legends. Statistical analyses were made using Students t test. Cerebellar granule neurons were grown and matured in-vitro in medium containing 25 mM KCl. When the method was changed compared to that containing 5. 6 mM KCl low KCl treatment., morphological changes including shrinkage of soma happened in some neurons at around 8 h after treatment as noted previously w8,15x. Mobile cleavage of HC-030031 Ac DEVD MCA, a of CPP32, steadily augmented at around 4 h and further improved until 8 h after low KCl therapy Fig. 1A.. Ac DEVD MCA cleavage activity was still observed 2-4 h later, when comprehensive apoptotic neuronal cell death was observed. On the other hand, when the medium was changed compared to that containing 25 mM KCl high KCl treatment., there was no change in Ac DEVD MCA cleavage activity. SNOW like activity to cleave Ac YVAD MCA was not enhanced after low KCl treatment at the time points examined, rather, there was slight reduction in this proteolytic activity. We also measured cellular MTT reduction activity and release of cellular LDH to culture medium, and compared the time dependent changes with these for Ac DEVD MCA cleavage, to examine the condition of the cells following the low KClrhigh KCl treatment.