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 Next generation therapeutic <http://www.ablynx.com/> antibodies Guy Hermans, PhD Project Leader Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: Losyev, Grigoriy [mailto:Grigoriy.Losyev@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 ----------------------------------------------------------------------- THIS E-MAIL MESSAGE IS INTENDED ONLY FOR THE USE OF THE INDIVIDUAL OR ENTITY TO WHICH IT IS ADDRESSED AND MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM DISCLOSURE. If the reader of this E-mail message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately at ablynx@ablynx.com. Thank you for your co-operation. -----------------------------------------------------------------------
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