CFDA-SE staining

Arnold Pizzey a.pizzey at ucl.ac.uk
Sat Jun 1 02:18:45 EST 2002


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
>
>

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	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 at ucl.ac.uk
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