Try coating your needle and syringe with the heparin to avoid clotting. Candace Enockson --On Mon, Dec 4, 2000 4:36 PM -0500 Bill Telford <telfordw@box-t.nih.gov> wrote: > > Hello all... > > My former postdoctoral mentor has this question regarding blood collection > from mice for flow cytometry. Any ideas? > > Thanks! > > Bill Telford > > _______________________ > Richard A. Miller (U. Michigan) asks... > > Got a question for you about mouse blood and flow cytometry. > We've been doing T cell subset on mouse tail blood for a while > now. The protocol involves heating the mouse under a light bulb; > then making a nick in the tail; then collecting 400 ul of blood into a > tube with a 10 ul drop of heparin in it; then washing with 600 ul of > PBS; then resuspending to a volume of 400 ul using BSS-BSA; > then aliquoting and adding antibodies. The flow rate has been 100 - > 200 events/second using our FacsCalibur; we typically count > 10000 events over 50 - 100 seconds. > We've now been doing the same thing but using cardiac blood > instead, and we get a much slower flow rate. The mice are > anesthetized (avertin); heart puncture using a 26 gauge needle > provides 600 - 1000 ul of cardiac blood. The needle is removed and > the blood is expressed into a tube containing 10 ul of heparin. We > then add 1.5 volumes of PBS; centrifuge; discard the supernatant; > add BSS to the original blood volume, and aliquot. But with this > method we get flow of only about 50 events/second, and > sometimes as low as 20 events/second. > I suspect a clotting problem; perhaps the damage to the heart > muscle, or perhaps by damage to the cells as they come into the > needle and syringe. The techs that do these bleedings tell me that > they do occasionally see evidence of slight clotting in the cardiac > samples, and I'm therefore suspicious that cells are being > sequestered or damaged during the blood collection process. > Do any of you have an idea of what's really going on here, or how to > solve it? > > Rich > _______________________ >
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