Help! How to prevent Neutrophil from iatrogenic activation? - (Oct/06/2006 )
Please give me any advices.
I am analyzing neutrophils with flouroscences-conjugated anti-CD18/41/61 antibodies by BD Flow cytometer. However, the neutrols from control mice were always activated?! I got a lot of positive anti-CD18/41/61 signals in control mice, especially anti-CD18!! My boss was mad and I was very sad.
So, anyone knows how to prevent iatrogenic activation of neutrophils, please please help me. Anything, any little tricks might help. Thank you very much.
ps. I use 3.8% sodium citrate as anti-coagulate and 0.5%(v/v) paraformaldehyde for fixation.
i m not quite sure how far i will be help full...
suggestion 1 - use 3.2%Citrate (dilute 1:9(blood)), look the attached article.
suggestion 2 -
use citrated blood directly for fax analysis (if u r using isolated neutrophils), so that u can be sure ur not messing up while doing ur experiment (n to avoid unnecessary steps). if this process does not work then u need to go for fixing with parafarmaldehyde.
use RT n on ice conditions for ur experiments (to catch the expected results somewhere rather repeating the exp with different variable)
thanks for reply, payeli.
one more concern about the paraformaldehyde, should i prepare 0.5% paraformaldehyde fresh or i can use a prepared stock solution? how long can i store the stock solution, 1 week or more??
i was using 1% parafarmaldehyde for monocyte surface receptor detection, i used to freeze 1%PFA at -20C and used for 4-5 times in 2 weeks.
i had expected results.
just want to remind u, y donot u try using direct blood for cd18 detection?