I work with RAW264.7 monocytes as model system, and came across the apoptosis time scale - how long quick is early apoptosis and how long the cell can remain in late stage?
it all depends on how you define those stages. I would expect that a majority of a culture can be induced to apoptosis (defined as AxV+/PI-) within just a few hours (3-4h post UV if that is your induced though it may depend on the method of induction). Changes in nuclear morphology (assessed by acridine orange and morphology) follow a little later as does cell shrinkage (assessed by FS v SS on flow). Late apoptosis (secondary necrosis defined as AxV+/PI+) will start to appear from a little later (perhaps 5-6h) but will peak at 12-16 h.
Dear Andrew,
Thanks for such detailed answer! Another question if you don't mind - why in some papers late apoptosis is combined with necrosis into one stage?
If they measure it by Anexin+PI+, usually is considered late apoptosis but it can also be necrosis. If necrotic, membranes are broken and PI is detected but it can also be detected phosphatidylserine, which is usually in the inner side of the membrane except in apoptosis when it translocates to the external site. When membranes are broken in necrosis PS from the inner site of the membrane can also be detected and you visualize it as A+/PI+ but instead of late apoptosis is necrosis.
Hi Olga, it is very difficult to give you a straight answer because it depends on a lot of factors, including the stimulus, and the cell line you are using. I would suggest you read the work from Markus Rehm, at the Royal College of Surgeons in Ireland, he has done some very detailed single cell imaging characterisation.
We can say in general that the initial phase of apoptosis, which involves activation of BCl-2 family proteins and depolarisation of the mitochondria is a relatively fast process, which happens within hours from the applied stimulus. Markus defined that the time required between depolarisation of the mitochondria and activation of the caspase cascade is usually around 30 min. The late phase after caspase activation, nuclear condensation and formation of the apoptotic bodies can take from as little as 3-4 hours to 24-48 hours.
If you induce apoptosis with high concentrations of TRAIL it is all executed within 4 h, if you use staurosporin v.s. cisplatin or proteasome inhibitors etc... usually it takes around 24 hours.
If you are using cells like MCF7, which are deficient of caspase 3 then the time doubles, and the cells take 48 hours to die.
This in general on a population level, because the cells are not synchronised and they start undergoing apoptosis at different times. If you start looking at single cells then you can start seeing some real time scales. Hope this helps, but I would really suggest to look at Markus's publications on the subject.
I agree that the terms early and late apoptosis are arbitrary and the answer would depend on what you want to learn.
You should find some interesting responses in the attached articles.
best regards
Robert
What is the significance of early apoptosis, late apoptosis and how we can justify.
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