Help! How to prevent Neutrophil from iatrogenic activation? - (Oct/06/2006 )

hi, Guys,

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.

hi,
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.

suggestion 3-
use RT n on ice conditions for ur experiments (to catch the expected results somewhere rather repeating the exp with different variable)

gud luk
sravan

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??

thanks

hi,
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?

gud luk