GFP and PE

Hi Flow Community,

I would appreciate any feedback on how flow cytometrists are objectively 
compensating GFP and PE and if they are encountering any issues.

We are using single color controls to set up the compensation. The GFP 
signal is robust (3-4 logs) and much of it spills over into the PE 
channel. Enough compensation to remove any spillover flattens out the 
signal and seems very subjective. Less compensation leaves GFP positive 
cells in the PE channel.

The cell surface mAb-PE signal can be high (2-3 logs) or small (1/2 ? 1 
log) depending on the targeted epitope. How can I be sure the single 
positively sorted PE cells are not just GFP positives in disguise due to 
incorrect compensation? We routinely perform sorts of ~98% purity with 
typical FITC & PE cell surface immunophenotyping. Cells with GFP only sort 
just fine as well.

We are using a BeckmanCoulter Altra with an argon (488nm) excitation and a 
550nm dichroic reflecting the GFP to a 510/21 band pass filter. The PE 
channel (PMT) is using a 625nm dichroic reflecting the PE to a 575/20 band 
pass filter.

My customers feel this should not be an issue.

Sincere regards,
 
Kent

Kent Claypool
Department of Carcinogenesis
University of Texas M.D. Anderson Cancer Center
Science Park - Research Division
1808 Park Road 1C
P.O. Box 389
Smithville, TX	78957

Tel. 512-237-9427
Fax: 512-237-9589