From: Joseph Webster (J.Webster@centenary.usyd.edu.AU)
Date: Thu Aug 20 1998 - 18:55:00 EST
Hi Mark, One potential cause is fragility of the cells. We find lots of variation in post-sort yield and/or viability between different cell types and preparation treatments. When you think about it, most of us would not be very useful after being thrown into a test tube at 10 meters per second... Others are much more equipped than I to comment on media etc., though one group here sorting mouse microglia had better viability results if they changed collection tubes much more often. I think they were changing after collecting 1mL or less, around 5 minutes. Joseph. At 10:10 19/08/98 +1000, Mark Cozens wrote: >Dear flowland > I am posting this message for a student in the department. He has >been experiencing trouble getting viable cultures after sorting. Sorting is >based on a two colour system. The viability has been checked (by 7AAD) at >every step of the procedure with the following results: > % viability 1: prior to sort 80% > 2: post sort after 3 days culture 1% > 3: post sort (unlabelled) 3 day culture 1% > 4: after 3 days culture, no sort 70% > >Cell in 2 and 3 were treated identically other than 3 being unlabelled for >the surface markers used in sorting. Cells in 4 were stained with the sort >antibodies (as a control) but not sorted. > >Sort conditions; > Sheath = ice cold PBS > Collecting into 15ml tube containing 1ml culture media (10% FCS) > Changing collection tube every 15 min (roughly 5ml total volume) > Cells suspended in cold culture media > >If any of this sounds familiar, or if anyone has any suggestion about >possible solutions, pleeeease email me. -- Joseph Webster Flow Cytometry Facility Centenary Institute
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