RE: bacteria sort on FACS Aria

Try to set up your sorter with 500nm beads as they are a better match in
their flow behaviour unless your cells are motile and active. Determine
your coincidence profile of particles as coincidence detection with
bacteria tends to fail. Depending on beam shaper and sheath flow
velocity / throughput and relative abundance of positives and negatives
your coincidence at 4000 events per second will be around 5 to 10%. Best
way to test it is to use non-green big beads and green small beads (for
example 3um and 500nm). In the coincident events you get big beads
showing a green fluorescence.

Good luck

Gerhard


-----Original Message-----
From: Taylor, Barbara Joan (NIH/NCI) [mailto:TaylorBa@mail.nih.gov] 
Sent: 08 June 2005 17:45
To: cyto-inbox
Subject: bacteria sort on FACS Aria


Fellow Sorters

I recently sorted bacteria with Qdots (ex 407nm, em 660/20) on the FACS
Aria.  There was a clear 10% "positive" population pre-sort. When I
reanalyzed the bugs in the positive sort tube, they were 100% negative -
no fluorescence at all.  I rechecked the drop delay with Accudrop and
then made a tube of 6 micron blank and fluorescent beads (ex 407 nm,
for the emission signal I removed the LP dichroic and replaced the
660/30 filter with a
450/40) and used the exact same sort setup as I had used with the
bacteria. These beads sorted perfectly with 99% purity on re-analysis.

We thought maybe the Qdots had detached (although the literature states
that they do not detach during sorting) so the sorted bacteria were
re-cultured; however they did not appear to be enriched for the protein
detected by the Qdots.

I have been using the Aria mostly for multi-color mouse lymphs and have
had no problems with sort purity.

We will be trying the bacteria again in a few weeks.

Any suggestions? 

As always, thanks

Barbara J Taylor
Facility Manager, FACS Core
CCR, NCI, NIH
Room 6008 Bldg 37
37 Convent Dr
Bethesda, Md 20892-4255
ph: 301.594.6892
fax: 301.496.8709
taylorba@mail.nih.gov