Problems with adjusting the flow cytometer - (Nov/24/2007 )

dear all,
I am working on the lymphoblastic cells (it grows in suspension) and I wanna do annexin V staining on my treated cells.
but the problem is how to adjust the flow cytometer.in the manual of the kit is written that I should have 3 control samples: normal,necrotic (for PI staining) and apoptotic
how can I prepare a control sample of apoptotis cells?
for necrotic cells it is suggested to use formaldehyde and methanol..!?
I thank you all in advance
regard
acute

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dear acute,
you could use staurosporine (STS). for time and concentration see sawa et al. nat med 1999, or cianide (maglione et al. mad 2006.

vittorio

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dear vittorio,
sorry for so much disturbance and thank you for ur helps,
unfortunately i cant find these 2 references.can u send me an exact reference and tell me where I can buy staurosporine?
as I told you in another forum ,I have so much problems with my cells,(they are growing tooooooo fast,use their medium(RPMI1640)rarely.....so it seems that there is a long way to reach the next step,flow cytometry I mean!!!
my cells donot even respond to my drug...toooooooo much problems....
thanks
bets regards
arash

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dear arash,
the exact refs are:
Maglione V, Cannella M, Gradini R, Cislaghi G, Squitieri F.
Huntingtin fragmentation and increased caspase 3, 8 and 9 activities in
lymphoblasts with heterozygous and homozygous Huntington's disease mutation.
Mech Ageing Dev. 2006 Feb;127(2):213-6.

Sawa A, Wiegand GW, Cooper J, Margolis RL, Sharp AH, Lawler JF Jr, Greenamyre
JT, Snyder SH, Ross CA.
Increased apoptosis of Huntington disease lymphoblasts associated with repeat
length-dependent mitochondrial depolarization.
Nat Med. 1999 Oct;5(10):1194-8.

staurosporine is SIGMA, cianide is fluka

if you have any problems don't esitate.

good luck

vittorio

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dear arash,
what is the serum concentration in rpmi ?you could use 30% serum in the medium, in addition you could use the multiwell plates with 24 or 6 well instead that flasks.

vittorio

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dear vittorio,
I use rpmi 1640 with 10% serum for my cells. another note is that I couldnot do MTT assay using the 96well plates.it seems that they grow not properly in this plates...is it better to use 24 or 6 well plates,then add it in the 96 well plate to read?
but what do u mean from using the plates instead of flasks?
regards
arash

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hi arash,
you can try with 96 multiwell plates. its not problem for mtt. however if you use the 24 multiwell plates, u can reduce the volume of the medium, so cells are more comunicantig and grow better than in the flasks. the serum concentration is good but when cells have some problems you could increase serum concentration in the medium.

regards
vittorio

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