Hello Debi, 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. Best regards, Arnold 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! >Debi > > _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 020-7679-6234 Fax: +44 020-7679-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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