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MTT - cell prolferation vs apoptosis (Dec/01/2009 )

Hi,

My stable cell lines grows slower than the parental cell line. I want to know where this reduced growth rate is due to reduction in celll prliferation or is it due to induction of apoptosis. Does this question seem logical or is it understaood that cells that grow sloer have high apoptosis? Sorry just wnat to know if i'm asking a silly question.

-SF_HK-

your cells could be decreasing the proliferation rate or dying (necrosis, apoptosis...). Just check on the microscope, if they are floating means that are dying. ;)

-blotted-

blotted on Dec 22 2009, 01:27 PM said:

Just check on the microscope, if they are floating means that are dying. :D

Not necessarily. they could have changed phenotype and become a suspension cell line (as opposed to attached cells) from the transfection or some other treatment during the stable cell line making. Some cell lines are composed of populations of both floating and attached cells. Dead cells will stain blue with trypan blue under a microscope, this is the easiest way to assess if floating cells are dead..

My stable cell lines grows slower than the parental cell line. I want to know where this reduced growth rate is due to reduction in celll prliferation or is it due to induction of apoptosis. Does this question seem logical or is it understaood that cells that grow sloer have high apoptosis? Sorry just wnat to know if i'm asking a silly question.

Your question can be answered by investigating whether there are increased levels of cell death in the stable cell line (e.g cell counts at time points counting populations of live and dead cells), but probably not by a proliferation assay which only look at live cell numbers. Assaying for caspase 3 will tell you if it is apoptosis.

MTT can be used for proliferation studies, but not for assessment of apoptosis, necrosis or autophagy. For each of these you need to assay for particular pathways, such as caspase 3 for apoptosis. Autophagy can be assessed by FACS using acridine orange.

-bob1-