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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