In article <318050A2.53D at aol.com>, <rjjensn at aol.com> wrote:
>I have been having trouble with Flurochrome labeling of Mouse LN
>cells. All labels downregulate L-selectin (CD62-L) except Hoechst,
>and our flow cytometer is unable to handle UV. The tags Ive tried are
>TRIRC, DiI, CMFDA, Pkh-26, and LDS If anyone can help with a dye that
>doesnt effect homing to LN I would be grateful.
We've used TRITC (I think that's what you meant) without l-selectin
downregulation, which leads me to wonder if your labeling conditions
are correct. Additionally, maximal stimulation of LNC (with PMA, for
example) results in full shedding after ~30min at 37 C, which is
pretty serious treatment. Just putting in your dye shouldn't do
this...
Make sure you're endotoxin-free! If you're getting mesenteric nodes,
be sure you don't nick the gut...
You can find a large number of in vivo studies in the
literature which use fluorescent LNC for homing studies, many of which
are visible with common FACS excitation/emission wavelengths.
Perhaps the most easy, cheapest dye is FITC. I refer you to:
Butcher EC; Weissman IL.
Direct fluorescent labeling of cells with fluorescein or rhodamine
isothiocyanate. I. Technical aspects.
Journal of Immunological Methods, 1980, 37(2):97-108.
Butcher EC; Scollay RG; Weissman IL.
Direct fluorescent labeling of cells with fluorescein or rhodamine
isothiocyanate. II. Potential application to studies of lymphocyte
migration and maturation.
Journal of Immunological Methods, 1980, 37(2):109-21.
As I say, there have been many more characterized since then. The real
question is what you expect to do with them. FITC is great for short
term (or even up to a few days if your just wondering about a general
trend in homing) in vivo homing. Recover the nodes, mash and run them
through FACS. Other dyes have better long term characteristics. If
you're only doing 5 minute assays, I think FITC is a good choice. You
can label LNC in 15-20 minutes, and if you're feeling adventurous, you
can even differentially label cells!
Be advised that 5 minutes is an extremely short period for this sort
of thing (homing of LNC), and that your cells must be in (very) good
condition (eg temp, suspension media, activation state, etc.). Also,
you will not distinguish between populations regulated at something
other than secondary adhesive interactions (ie, the transient firm
adhesion which follows rolling) or more likely initial interactions
(rolling)...
Best of luck,
Aaron
--
"Nothing more is needed to destroy a man, than the conviction that his
life's work is useless." -Antonin Artaud
erawtech at leland.stanford.edu (R. Aaron Warnock)
