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isoflurane and FACS analysis of peripheral blood? - my cells look terrible everytime I use isoflurane to anaesthetize (May/16/2009 )

whenever i obtain peripheral blood from mice (black 6) after isoflurane anaesthesia my FACS results are terrible. most cells look dead (PI exclusion) and no granulocytes are seen.

i have to use an inhalant anaesthetic because my collaborator is taking the brain for electrophysiological recordings.
we anaesthetise the mouse in 2% isoflurane for a few seconds, and then we perform decapitation. I obtain the peripheral blood in EDTA (0.5%) pretreated blood, perform a RBC lysis (BD Pharmalyse) for 5 minutes, and wash the cells with PBS at 1200rpm for 6 minutes.

when i try the protocol on mice without isoflurane (but CO2 for example) the blood looks perfect in FACS - all the cell populations are seen.

is there anything you recommend? does anyone know how isoflurane is effecting blood?

many thanks

-euglena-

I would suggest you to look for other reasons. If isoflurane is this toxic, it will not be approved for any animal use.

-genehunter-

According to a quick Pubmed search, there's some evidence that halothane anesthesia can induce apoptosis of immune cells. A few papers you may want to take a look at:
Vet Res Commun. 2008 Dec;32(8):619-26
J Appl Genet. 2005;46(3):319-24
Int J Immunopharmacol. 1997 Nov-Dec;19(11-12):699-707

Another option for anesthesia could be intraperotineal avertin. There's precedent for using cells from rats sacrificed this way for EP. Check out:
Nature Protocols 2007 Vol. 2 No. 1 pp. 152-160

Good luck

-gfischer-

Is it not possible to draw a blood sample before anesthesia? Or is this the purpose of the study?
One thing I found helpful is diluting the blood sample 1:1 with PBS to increase fluidity of sample.

-Stephan-

We use isofluorane to anesthetize mice then do retroorbital eyebleeds to obtain blood for our flow cytometry experiments. I suppose this is different that what you are doing though.

-Matt105-

Isolflurane is used in pediatrics for anesthesia and is much more forgiving than Halothane in my experience. They are certainly not the same drug in reference to that apoptosis article. I know this sounds silly but make sure your PBS is 1X. Last week our lab's undergrad was prepping our EDTA/PBS solution that kept yielding horrible viabilities on the sorter. Sure enough she hadn't noticed the small 10X on the container.

-rich636-