FW: CFSE

Just to clarify the email I sent yesterday...

We label the PBLs with CFSE (5uM concentration) for 10-15mins 37C. Then
wash 3x with serum-rich media, then incubate o/n at 37C to allow leakage
of unbound CFSE etc.

Despite the above we are still seeing rapid loss of CFSE.

Thanks to everyone who has sent advice

Matthew Henry


-----Original Message-----
From: Matthew Henry 
Sent: 25 May 2004 11:13
To: cyto-inbox
Subject: CFSE

Hi Flowers

Someone has contacted me regarding CFSE labelling of human PBLs. I've
exhausted my minimal supply of flow knowledge...

The problem they keep encountering is that the PBLs lose CFSE very
rapidly (~18hours). 

Briefly they label fresh human PBLs with 5uM CFSE, incubate o/n @37C to
allow equilibration of excess/unbound CFSE. They check the fluorescence
of the labelled cells by flow (BD FACScan) after labelling (signal in
the 4th decade) and the next day following equilibration (signal in the
1st decade). The CFSE labelled PBLs are losing their CFSE... I don't
understand this.

Has anyone else seen this phenomenon?

Matthew Henry

CD45 Group
Edward Jenner Institute for Vaccine Research
Reading
UK