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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunology forum and cytometry forum
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FACS sorting normal donor B-cells
Posted by: supremo (IP Hidden, New member, 1)
Date: January 9, 2007 11:36PM
Hi all,
I'm having trouble FACS sorting normal/ donor B-cells from peripheral blood samples (in EDTA). Sorting preparations include: removal of plasma by spinning 400g. Then Ficoll/ Lymphocyte separation prep., followed by washing the buffy coat with PBS twice (First at 100g for 20 mins, then 400g for 20 mins). THe washing step supposed to remove most of the platelet contamination. The sample was then stained with CD19PE/ CD5 FITC/ and 7AAD (viability) - with the goal to collect pure population of viable CD19+ Bcells and CD5+ T-cells. The main problem is that, despite all that, I cant seem to get high enough percentage (+/- 2% out of the total population) of B and T-cells to FACS sort efficiently. There would still be debris ie. a large percentage of low FCS and low SSC population that slows down the sort considerably. I tried increasing the intial volume of peripheral blood (20ml), but doesnt help too. So the problems remain despite increasing number of lymphocytes pre-sorting. Can anybody help me??? Please ask me more if my explanation's not clear enough...
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