Biotin for red blood cells labeling

Guy Hermans Guy.Hermans at ablynx.com
Tue Nov 8 02:37:02 EST 2005


Hi Grigoriy,
 
I fail to see why getting total cell population labeling is required to
determine that populaqtion's half-life. Better get high intensity staining
of a subset with a reagent which won't interfere with turnover than getting
total labeling with something which might change the observed parameter -
and I'm not so sure biotin is the best choice in that respect (I may be
wrong here).
 
I'd recommend labeling with CFSE if that's possible, or ex-vivo label packed
cells with CFSE and reintroduce them via tail vein. This label is
"invisible" biochemically on the outside of the cells and is therefore less
likely to change halflife. Also, it allows direct measurement of
fluorescence, so any additional time spent during labeling in this
alternative protocol would be regained during sampling/analysis.
 
In any case: I'd suggest introducing the label (biotin or otherwise)
intravenously (tail vein) to get better/faster distribution than
retroorbital.
 
Good luck,
 
Guy
 
 



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-----Original Message-----
From: Losyev, Grigoriy [mailto:Grigoriy.Losyev at childrens.harvard.edu] 
Sent: Friday, November 04, 2005 6:38 PM
To: cyto-inbox
Subject: Biotin for red blood cells labeling



Dear Flowers

 

Our current project is labeling RBCs with 1.5 mg NHS PEO4 biotin injected
retro-orbitally in 3 doses over 24 hours and then watching RBC aging through
time. 

After labeling RBCs in mouse's blood stream we have drawn blood twice a week
and stain RBCs with Alexa 488 SA.  Then we pass cells trough flowcytometer.
We are measuring percent of stained cells. During the time course it goes
down.

 

My question is related to biotin quality. We never were able to label 90% of
RBC at the initial point. Does it depend on biotin? Is there a better grade
for biotin?

Could somebody suggest what could improve initial labeling of RBCs?

 

Thanks 

 

 

Grigoriy Losyev

Division of Hematology/Oncology
Flow Cytometry

Children's Hospital Boston

Karp Family Research Facilities, 08010

300 Longwood Ave 
Boston MA 02115 
Phone- 617.919.2127
Fax- 617.730.0934 

 



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