 |
 |
 |
 |
 |
>As for quenching, perhaps someone can address that question. The
fluorescence
generated from the phenol red alone will affect your results and
the ability to
differentiate populations with different staining intensities.
REPLY:
Phenol red is indeed a problem. When lymphocytes are being
labelled, it appears that adequate washing of the cells is enough to
remove the "autofluorescence-like" problem of phenol red. In
contrast, myeloid cells present a particular problem, especially
when they have been activated.
In order to prevent this problem, we use phenol red-free DMEM (from
Gibco-BRL/Life Technologies). It is possible to use their
"standard" phenol red-free DMEM (cat. no. 11880), but this is "low
glucose". We prefer to have "high glucose" medium - because of the
variety of cell types with which we work - and also medium
supplemented with bicarbonate and 25mM HEPES, because we no longer
have any visual control over pH changes in the media.
If we order medium 11880, we add glucose (to 4500mg/L) and HEPES
(to 25mM), as well as Glutamax as a source of "stable" glutamine.
Alternatively, we place a "special" order for a modification of the
"high glucose" DMEM containing HEPES (cat. no. 42430 or 32430) that
it should be phenol red-free (such an order takes about 6 weeks to
prepare and deliver).
Should growing cells in phenol red-free medium pose a problem
concerning the lack of visual control over the pH, it is possible to
set up "control cultures" in which the cells are placed in
conventional phenol red-containing medium; this will prove a useful
indicator of pH changes in the cultures.
We have been using phenol red-free medium with cultures of porcine
and bovine leukocytes, including bone marrow cells, over the past 6
years without any apparent problems.
Kenneth McCullough.
Cell Biology,
Institute of Virology and Immunoprophylaxis,
CH-3147 Mittelhausern,
Switzerland
Tel. (+41) 31 848 9361
Fax (+41) 31 848 9222
email: kenneth.mccullough@ivi.admin.ch
|
|
|
|
|
|
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