How to reduce the background of Annexin V staining? - (Apr/18/2006 )

Hi everybody,
I am doing flow cytometry, 2-color staining: PI and Annexin V. PI staining is ok, but with Annexin V staining, the background is quite high, and always I have to increase the compensation tp 30-40%.
I would like to ask how to decrease the backgound for annexin V staining?
I use 0.5x10 (6) cells for each time detecting.
I tried to increase the volume of Wash Buffer after staining, but it just reduced slightly.
Thanks so much for your help!

Hi ,
Iam also doing same experiment i think you can minimise its by avoiding to spining cells at high rpm/g and gentelly dispersed its in the wash buffers.
Like never spin more than 1500rpm in table top centrifuge for 5 min. and disspersed pellets smoothly and all other precaution you shuld possible to take its.
for further quairy you can contact me by this forum and also bye directly mail me at awadh507@gmail.com
all the best.

Thank you so much for your willing,
Actually, I tried to increase with volume of Wash buffer after staining, and the background decreased, but not significantly. And still the compensation is high (40-45%)
I wonder should I reduce the CaCl2 concentration? Does it affect background of Annexin V?
In previous expreriments, I used 5mM CaCl2. If yes, which concentration should I used?

you can reduces its and check at different molarity like 4,3 and 2.5 i thinks its give you idea about which one good for your experiment . But dont increase volume more than 300ul.
all the best and good luck.
awadh

Thank you so much for your willing,
Actually, I tried to increase with volume of Wash buffer after staining, and the background decreased, but not significantly. And still the compensation is high (40-45%)
I wonder should I reduce the CaCl2 concentration? Does it affect background of Annexin V?
In previous expreriments, I used 5mM CaCl2. If yes, which concentration should I used?

In the first time, I did not wash, and the background is very high!
The next time, I washed with 3ml and 4ml Wash buffer, and it was better.
I stained the cell in 200ul Buffer containing Annexin V and PI.
Hence, complicated, hix hix...
Another thing, according to some papers I read, they presented the high background of Annexin V, and just focusing on the difference between Control sample and Stimulated samples. So, I wonder what is the meaning of low background?

I use PI staining and Annexin V [it's a kit called "Annexin-V-Fluos"] - I don't know what the fluorophore on the annexin is but I think it's FITC - but there's a large amount of overlap in the spectral emission from them, so you need a lot of compensation in the green channel [Annexin V channel] to seperate them out.

Background: For adherent cells that need to be trypsinised to detach them, I find that you need to treat the cells very, very gently. My background was ~15% initially, but once I started being more gentle about the trypsinisation and handling of the cells, it fell to <5%, allowed me to see the real difference between the apoptotic cells and the healthy cells.