Hi Phil, My suggestion to you is to eliminate the lysis step all together. You could eliminate the rbc population by overlaying mouse whole blood with Accupaque and use the gradient seperation method. If you can obtain ~3 mls of mouse whole blood dilute it 1:1 with PBS, then overlay the diluted blood with 3 mls of Accupaque, centrifuge at 1600 rpm for 30 minutes at room temperature with no brake. Remove interface, then bring final volume to 12mls with PBS and centrifuge at 1000 rpm for 7 minutes. Resuspend pellet in 10 mls PBS, count viable cells and repeat centrifuge step. At this point you can resuspend cells in staining buffer in preparation to block and stain. Ultimately you have mouse blood without rbc's, platelets, and neutrophils. The viability is generally high and your cells are live and not fixed. Also, I suggest collecting the mouse blood in an EDTA-anticoagulate tube. I hope this method is useful and that I appropriately addressed your issue. Diane R. Connor Sr. Scientist GlaxoSmithKline Protein Agents & Human Gene Therapy Diane_R_Connor@gsk.com Phone: 1-610-270-7650 Fax: 1-610-270-5381Received on Thu Sep 9 16:18:00 2004
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