Hei,
I analyzed caspase3/7 activation (using the Kit from Promega) 24, 48, 72 and 96 hours after treatment (se figure pdf). Caspase activation has increased 24 hours after treatment, is highest after 48 hours and then decreases again.
I was wondering when these two caspases are activated upon apoptosis. Are they activated at an early time point? Does the activation decrease as apoptosis progresses?
When this capases are activated, the cells are in the final of apoptosis.
The decrease of caspase around the time occurs because all the cells which suffer this process died, then the others which not will maintain the normal levels of these caspases.
I can say that one can kill cells via apoptosis within 6-12 hours which clearly suggest activation of caspases may occur even in earlier time points. Once initiated caspase activation is a very quick process as it does not require transcription/translation machinery. They are activated via proteolytic cleavage leading to a cascade of activation and it is usually difficult to track the time course. Even though caspase-3 is an effector which gets activated in late part of apoptosis, I don't think there can be a timecourse of days between the activation of caspase-8 -9 or -10 and -3.
The decrease you have detected may be due to the higher number of cell deaths. Do you know if your cells are alive there or not? Have you corrected your results according to the protein concentration in your samples (as samples with fewer cells in it would have less protein)?
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