I also have my best experience with the succinimidylester of
CFDA. The trick of internal cleaving for activation is also
a nice way to exclude dead cells from analysis. The fairly
gentle reaction of the succinimide group has also been used
by others to biotinylate whole cells which would give access
to a variety of secondary labels to suit the calcein-AM. For
some articles on biotinylation see below.
Already with the best wishes for a happy and peaceful
Christmas to all of you
Gerhard
-------------------------
Title: Methodologic considerations for the use of canine in vivo aged
biotinylated erythrocytes to study RBC senescence.
Author: Christian J A; Rebar A H; Boon G D; Low P S
Source: Exp-Hematol 1996 Jan, VOL: 24 (1), P: 82-8, X.
Year: 1996
Company: Department of Veterinary Pathobiology, Purdue University, West
Lafayette, IN 47907-1243, USA.
----------------------
Title: BIOTINYLATION OF CELL-SURFACE MHC MOLECULES - A COMPLEMENTARY TOOL FOR
THE STUDY OF MHC CLASS-II POLYMORPHISM IN CATTLE
Author: SCHUBERTH HJ; KROELL A; LEIBOLD W
Source: JOURNAL OF IMMUNOLOGICAL METHODS 1996 189 N1 89
Year: 1996
Company: SCH VET MED,IMMUNOL UNIT,BISCHOFSCHOLER DAMM 15
Country: D-30173 HANNOVER GERMANY
-------------------------
Title: Studies with biotinylated RBC: (1) use of flow cytometry to determine
posttranfusion survival and (2) isolation using streptavidin conjugated magnetic
beads
Author: Russo, Vincenzo; Barker-Gear, Robin; Gates, Robert; Franco, Robert
Source: Adv. Exp. Med. Biol. 1992 326 Use of Resealed Erythrocytes as Carriers
and Bioreactors 101-7
Year: 1992
Abstract Methods are reported for the quantitation and isolation of biotinylated
red blood cells (B-RBC). The first method is for detn. of posttransfusional
survival of rabbit RBC by flow cytometry. The survival of B-RBC was measured
using both fresh and paraformaldehyde-fixed cells with similar results. The
posttransfusion survival of rabbit RBC measured in this way was normal. There
was no indication of increased cell destruction due to antibodies directed
against B-RBC and no evidence for loss of biotin from circulating cells. The
second methodol. is for the isolation of B-RBC from blood with
streptavidin-coated magnetic beads. At least eighty percent of pos. cells were
recovered with very few false positives. Both methods may be helpful in the
study of resealed erythrocytes.
-----------------------------
Title: In vivo biotinylation demonstrates that reticulated platelets are the
youngest platelets in circulation.
Author: Ault-K-A; Knowles-C.
Source: Exp-Hematol 1995 Aug, VOL: 23 (9), P: 996-1001, ISSN: 0301-472X.
Year: 1995
Company: Maine Medical Center Research Institute, South Portland 04106,
USA.
In both mice and humans, a subset of platelets can be identified that shows
increased labeling with nucleic acid-specific fluorescent dyes, such as thiazole
orange. Termed "reticulated platelets, " they have been postulated to be
platelets that have recently entered the circulation. Their numbers appear to
reflect the rate of new platelet production in a number of clinical and
experimental situations. To determine whether reticulated platelets really are
the youngest platelets in circulation and to estimate the length of time that
they are identifiable after entering the circulation, we have employed a
technique of "in vivo biotinylation" in mice that labels the entire cohort of
circulating cells with covalently bound biotin. Blood samples can then be
double-labeled with fluorescent avidin derivatives and thiazole orange,
permitting correlated measurement of both surface biotin content and nucleic
acid content. The biotinylation occurs rapidly, is complete within 30 minutes,
is stable for several days, and does not appear to alter platelet function. The
results show that within 24 hours after in vivo biotinylation, platelets appear
in the circulation with decreased levels of biotinylation and that these are the
reticulated platelets. The estimated lifespan of reticulated platelets is 1.8
days, and the lifespan of all platelets by this method is 4.5 days, which is in
agreement with estimates made by other methods. Author.
______________________________ Reply Separator _________________________________
Subject: Gap junction flux measurements
Author: R.Nordon@unsw.edu.au at INTERNET
Date: 12/12/97 15:01
A number of vital stains bind to cytoplasmic protein and may be more
suitable for your application. Some emit in the green region (CFDA, SE
C1157) or in the red region (SNARF-1 C-6826 or CellTracker orange C-2927). I
have only had experience with CFDA, SE which I have used to track cell
division. About 1/10 of the dye binds to intracellular protein whilst most
of the unbound dye leaks out in the first 24 hours, with very little leaking
of stain thereafter. See British J. of Haematology 1997; 98:528-539 for details.
Robert Nordon
Post Doctoral Fellow
Graduate School of Biomedical Engineering
University of New South Wales
Sydney, 2052
Tel. no. 61-2-9385-3906
Fax 61-2-9663-2108
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:50:25 EST