Prevent Neutrophil Cells from Iatrogenic Activation

[Kellee]

Prevent Neutrophil Cells from Iatrogenic Activation:
Hi, Im currently analyzing neutrophils with flouroscences-conjugated anti-CD18/41/61 antibodies by BD Flow cytometer. However, the neutrophils from control mice are always activated!

I use 3.8% sodium citrate as anti-coagulate and 0.5%(v/v) paraformaldehyde for fixation.

I received a lot of positive anti-CD18/41/61 signals in control mice, especially anti-CD18 and my boss was mad and I was very sad .

If anyone knows how to prevent iatrogenic activation of neutrophils, please please help me. Anything, any little tricks might help. Thank you very much.

[postdock]

3 suggestions for you:

1) Use 3.2%Citrate (dilute 1:9(blood)), look the attached article.

2) Use citrated blood directly for fax analysis (if you are using isolated neutrophils), so that u can be sure your 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.

3) use RT and on ice conditions for your experiments (to catch the expected results somewhere rather repeating the experiment with different variables)

[DNA_science]

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.

have you try using direct blood for cd18 detection?