One of our researchers has the same problem. I have been trying to get them to infuse and harvest the next day instead of several weeks. I am still working on that. I am questioning if the cells are still alive or if they are no longer green. How do we tell??? I also would like any hints/suggestions from those who have tried this. Jim Houston > ---------- > From: deborah foss > Sent: Thursday, May 30, 2002 4:53 PM > To: Cytometry Mailing List > Subject: CFDA-SE staining > > > > -- > I just found this list while searching for reasons that the CFDA-SE > staining is not working in our Laboratory. What we are trying to do > is stain Peripheral Blood MC and inject them into the same strain of > mouse. The CFDA-SE staining would be the way to tell the donor and > host cells apart. We are using Balb-c mice. We sacrifice the donors, > spin and wash the PBMCs, lyse the cells, wash again, count them, > stain them with from 1uM to 10uM of the vibrant CFDA-SE made up as > the package directs. We have tried incubating from 10 to 15 minutes, > we have tried stopping the reaction as the directions said (wash, and > 30 more minutes in the water bath). We have tried using 5%FCS to end > the reaction. The cells are stained, we check on the Facs, they are > all the way to the right on the log scale. > Then we put them in the animal (IV), from 1 million to 5 million. > When we take the tissues from the animals, (24 or 48 hours later), we > don't see any staining on analysis with the Facs. We have checked > both the spleen and the thymus. To check out if it was a problem with > the staining, I irradiated the latest group of mice, 600r, 2 hours > prior to the transfer. Still no green cells. > I know that we are looking for a small population of cells, but I > figure we have to be missing something. > We are not looking for cell division, just adoptive transfer. Help! > Debi > >
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