J.Webster at centenary.usyd.edu.AU
Thu Aug 20 18:55:00 EST 1998
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.
At 10:10 19/08/98 +1000, Mark Cozens wrote:
> 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.
> 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.
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