OVA acute asthma mouse model - mouse model stopped working! (Jun/09/2010 )

Hi y'all

I have a whole bunch of questions, since I am neither a trained immunologist and only recently involved in this mouse model.
Everything seems to be working fine in the regular mouse room (open caging and soft paper bedding) with bought wt mice and a different k.o. strain (all C57BL6/J)...now everything stopped working when we switched to the special barrier/ultra clean room for out new k.o. mice using them and their litter mates, means I don't see any inflammation after challenge- well so far no increased cell number in the BALF and only enhanced Macrophage number in the BALF in the chronic model.

I have a couple of things which changed and might be the issue and would like everyone's feedback:

1-I had to switch lot# of the OVA I use, but if I recall right it still worked in the "not so clean room" for me and Sigma doesn't sell my original lot anymore. I will post the lot numbers once I am back in the lab.- did anyone have similar issues??

2- clean room has different bedding...not the soft paper one but rather the hard granular one (I do not know right now the professional terminology for it)-issue?

3- of course now my new knock out mice and litter mates breeze filtered air and are all enclosed

all in all I don't get almost any immunological response at all..means my PBS controls are like my OVA challenged ones and this is true for my wt controls as well as my k.o. ones except for outliers?? (2 in 12??)

differences beyond housing to experiments before: I use N6+ litter mate controls to k.o. (bred back to C57BL6/J background, should be 90+ at this stage)

I don't understand!!!

protocol which worked so far is:
day 0 and 12 IP with OVA/Alumn
day 28, 30, 32 intranasal challenge
the amounts are the same as before so can't be it!

Beyond these problems I am also wondering why I don't see more eosinophils than macrophages in my regular model (open caging)...I see definitely enhanced numbers but not more than macrophages. This is even more so in the clean room in my chronic model-not working.
What does that mean?
I know some people see enhanced eosinophils but macrophages are still predominant in their OVA mouse model- it seems to be determined by their facility- too clean? too dirty?
I know a lot of neutrophils would be worrisome since an indication for an actual infection. No problems with that.
I was told it is what it is-but I am still trying to understand and optimize my model.

Beyond-that doesn't solve the problem of no difference for PBS versus OVA in my ko and wt mice in the clean room!!!!

Any help or explanation or experience reports (OVA lots which work? etc) welcome!!!

I think your issue may lie in the different OVA lots you are using. Compare your Product Data/Analysis Sheet for old and new OVA and see if differences in e.g., endotoxin amounts are present - my guess is that the endotoxin levels are not the same and lower in your newer lot. Hope you find the answer. Such setback can be very frustrating.