a.pizzey at ucl.ac.uk
Sat Jun 1 02:18:45 EST 2002
We did a similar experiment some years ago looking at engraftment of human
CD34+ cells into Balb-cs. We used CFSE to vital stain the CD34s, I am
replying from home so I don't have the relevant lab books to hand but, if I
recall correctly we labelled the cells with CFSE then sorted them to obtain
a tightly defined fluorescence peak before injection. We reanalysed bone
marrow from the recipients and recovered a small CFSE population. I
remember that I had to run a LOT of cells to see the positive peak. Email
me next week and I will hunt out the protocol.
At 17:53 30/05/02 -0400, you wrote:
>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!
Arnold Richard Pizzey
Department of Haematology
Royal Free and University College London Medical School
98 Chenies Mews
London WC1E 6HX
voice: +44 020-7679-6234
Fax: +44 020-7679-6222
email: a.pizzey at ucl.ac.uk
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