FACS- Propidium iodide experiments

[srilathabio]

I would like to do cellcycle analysis by Propidium iodide-FACS.want to show apoptosis in drug treated jurkat cells compare to untreated.but sub G1 mean is not incresing and G1/G0 mean also same.please help any expert in this field.thanks in advance.

[Warthaug]

Propridium iodide only enters apoptotic cells that have begun to loose their membrane integridty - something which occurs very late in the apoptotic process (technically, after its over). If you're not stimulating a cell long enough, you will not see PI staining; even if the cell is in advanced stages of apoptosis. You may want to try a marker of an earlier stage of apoptotis, like annexinV, or incubate your cells for longer.

Bryan

[srilathabio]

Thanks warthaug for your suggestion.this time i decresed the cell no and incubated with drug more time.and kept positive control 100mM H202.tommorow i will see results.i will let you know.is there any other positive controls? h202 100mM is it ok?

[srilathabio]

Sorry for late response.Iam unable to attch histogram figures here,but please tell me in control and drug treated no of events should be same?or it can change?
for example in control and drug treated histogram statistics are like following,
Control:
ALL-8584
sub G1 region-378
G1 region-5292
S region-1533
G2 region-1139

25ug/ml drug treated:
ALL-6132
sub G1 region-689
G1 region-3423
S region-900
G2 region-840

50ug/ml drug treated:
ALL-7559
sub G1 region-3256
G1 region-2262
S region-1356
G2 region-94


100ug/ml drug treated:
ALL-4891
sub G1 region-4088
G1 region-2629
S region-133
G2 region-4

Is this data correct? where is error?
please suggest me.thnaks in advance.

[wr_1a]

thanks