isoflurane and FACS analysis of peripheral blood? - my cells look terrible everytime I use isoflurane to anaesthetize (May/17/2009 )
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?
I would suggest you to look for other reasons. If isoflurane is this toxic, it will not be approved for any animal use.
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
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.
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.
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.