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You asked for a method to stain cells for flow which will distinguish
between live and dead cells when co-stained with a PE conjugated Ab.
We have used eosin Y (aka tetrabromofluorescein) for live/dead
discrimination in yeast cells. This method selectively stains non-viable
cells without affecting the cellular viability of competent cells. The
method entails resuspending washed cells in 0.01% eosin Y/0.15M NaCl and
incubating at 25 degrees C for 10 minutes. Wash the cells and you are
ready to go.
If you speak nicely to the people in your nearest histo lab, they might be
able to provide you with the eosin (that's how we obtained ours!).
We have not carried out any dual staining with labelled antibody but it
should be easy enough to incorporate this into the method. Eosin Y
fluoresces green with an emission max between 515 and 518nm, so you can
pick up the signal using whichever detector you normally use for FITC.
For more details, see Costantino, P. J. et al Cytometry 19:370-375. (1995).
Good luck
Regards,
Dianne Budd
Room 308:206 Tel: (09) 351 7177 Fax: (09) 351 2342
Int: 619 351 7177 619 351 2342
School of Biomedical Sciences
Curtin University of Technology
GPO Box U1987 Perth 6001 WA
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web