RE: FACS DiVa question

This sort of strange thing has happened to me three times while sorting.  Once, I had a
DAPI negative gate for live cells set for a five-color sort, and upon analysis of the
sorted cells, the whole population disappeared to the bottom of the axis, as you've
described.  No other parameter was affected, and the sort purity was fine, so I ignored
it as one of those "digital issues".  Another time, I did a similar sort, and the PE-Cy5
bright (4th decade) cells looked completely negative (1st decade) upon analysis of the
sorted cells, but all the other parameters sorted as expected.	The stem cell antibody
that was used with the PE-Cy5 fluorochrome correlated with the other markers and only
positive cells would have sorted based on the analysis of the other parameters. (Plus,
the next sample sorted with the same staining pattern looked fine after sorting.)  Again,
the purity was fine based on the analysis of the other parameters.  

Both times we put the presort sample back on and did a second tube analysis, and the
parameters looked as they should.  I showed the data to my service engineer, but he had
no explanation or fix.	The problem was completely random, and the parameter affected was
different each time.  Since then, we have upgraded the DiVa software to version 4.0, and
we haven't had anymore issues related to that.

Does anyone know what is going on with that?

Jodi 

-----Original Message-----
From: Joseph Webster [mailto:J.Webster@centenary.usyd.edu.au]
Sent: Monday, March 01, 2004 11:00 PM
To: cyto-inbox
Subject: FACS DiVa question


Dear All,
Has anyone seen the negative cells disappear off the bottom of the scale?

We have a 3-laser DiVa, 488+600(dye)+UV.
We have observed this using DAPI as a "dead cell" label excited by
the third (UV) beam, but have also seen it with very bright PE from
the primary laser.

Users here commonly run with a DAPI (or PI) concentration that puts
dead cells at the top of the scale, sometimes over the top.
We (usually) see the "live" cells in the bottom decade, a smear in
the middle, and a peak of "dead" cells at the top.

We recently noticed that the "live" peak is sometimes dropping off
the bottom!
The middle and high intensity signals don't seem to change, only the
negative.
More tests to do, maybe the proportions are changing also...

The effect is dependent on sample flow rate &/or differential
pressure; it has been made to appear and disappear by winding the
differential pressure up and down.

It only shows up when there is signal at or near the top of the scale.
Comparing two tubes in the same experiment, one with moderate intensity
PE, the other with very bright PE, we could make the negatives drop
off the bottom when there was bright PE present, but in the tube with
lower PE it would not happen.

It may be affected by alignment or focus, don't know yet.

Maybe someone with DiVa experience can tell us what's happening &/or
how to stop it.
Don't know if other DSP-based systems show anything similar, I have
not seen it in any of our analogue instruments...

There's always something new to learn in this game!
	 Joseph.

--
Joseph Webster, Flow Cytometry Facility,
Centenary Institute
Locked Bag No.6, Newtown, NSW 2042, AUSTRALIA.
Ph: 61-2-9565-6110	Fax: 61-2-9565-6101
e-mail: J.Webster@centenary.usyd.edu.au 

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