Very large lymphocytes

Hi. We have a situation where a subset of cultured T cells appear to 
get so large when stimulated that they are off scale on standard 
FSC/SSc gates acquired on a BD LSR2. The voltages are set to 
accommodate non-stimulated lymphocytes and are not abnormally high 
and the cells are not fixed.  In our experience, blasting T cells sit 
approximately 2-3 times further out on the FSC than non-stimulated 
cells and a bit higher on the SSC, but up to a third of our cells in 
the activated culture are so large that they sit on the junction of 
the axis at the top right of a normal FSC/SSC plot, whereas another 
third or so sit on the far right border. Dead cells have been 
excluded by 7aad and a dump channel to remove autofluorescent cells, 
whilst doublets are removed by standard H vs W for both FSC and SSC. 
Given what we thought were fairly rigid controls, should be trust 
these on-axis events? On the face of it, the FSC suggests they are as 
much as 10 larger than normal, but looking at these cells down a 
microscope, they don't appear that large. Any thoughts? If they're 
not real, how do we prove it and/or exclude them?

Stuart

-- 
Stuart Berzins Ph.D,
Research Fellow, Godfrey Laboratory,
Department of Microbiology and Immunology,
The University of Melbourne,
Parkville 3010,
AUSTRALIA.
email: berzins@unimelb.edu.au
Ph:	+61-3-8344-5704
Fax:   +61-3-9347-1540
Mobile: 0427 849 123