Toxicity of CFSE staining

From: Max Warncke <max.warncke@uniklinik-freiburg.de>
Date: Wed Apr 06 2005 - 04:53:49 EST
Hi,
when I stain murine lymphocytes with 5然 CFSE (or more) I have a
substanstial loss of vitality compared to 0,5然 or nostain samples. If this
is normal, what is the usual percentage of dead/apoptotic cells after 5然
CFSE staining. If not could this be a problem of the staining protocol?

After the staining I am using the CFSE labeled splenocytes for "in vivo
cytoxicity assay", where I inject a 0,5然 and 5然 CFSE stained population in
a 1:1 ratio. If the 5然 cells have a reduction in vitality it is very
difficult to inject the same amount of vital 5然 cells. Sometimes my 5然
population is 20% lower than the 0,5然.

Thank you very much

Max




Protocol:

CFSE staining:

Ery lysis with 9ml H20 + 1ml 10x PBS, wash with 1xPBS
2x10E7 cells / ml + the same volume of 10然 CFSE solution 10min 37蚓
stop with 10ml protein containing media
wash 2x


"in vivo cytoxicity assay":

inject 1x10E7 0,5然 and 5然 labeled differentially peptide loaded cells i.v.
after 24h analyse blood
Received on Wed Apr 6 11:58:01 2005

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