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.
>rjjensn at avalon.net
I'm not sure what Pkh-26 is, but if it's one of those fluorochromes
from Zynaxis, then you should be able to use the PKH2-GL version of
the dye. While I haven't done this in around 4 or 5 years, I do
recall having checked lymph nodes for transferred cells and they
went there. However, I was more interested in the fate of B cells
in the spleen so I hadn't checked for MEL-14 staining prior to the
transfer.
I had also consulted the company a number or times
since they were just getting started and were extremely eager to
help (now that Sigma is their distributor, I'm not sure if this is
still the case). One suggestion was to decrease the labeling time
to one minute and titrate the dye, rather than use the suggested
labeling time. The original labeling time not only affected the
phenotypes but also the viability of my cells (in fact, it killed
most of them) while the new time worked out well.
If you decide to try this, feel free to contact me by email and I'll
hunt down the labeling conditions I used (cell and dye concentrations,
etc.)
Stacy
