>-- >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 Your protocol basically sounds right but I have a few of questions... * what concentration are your cells at when you stain them with CFSE? It has been suggested that CFSE is toxic and that this effect is particularly pronounced with low cell numbers. * how many cells are you collecting from your spleen samples? (I would not expect that you would see a significant number of donor cells in the thymus) * have you tried labelling spleen or LN cells following the same procedure? You should see a significant population of CFSE+ cells if you transfer 1-5 million spleen or LN cells. This would at least tell you that your staining and organ processing etc is working... * what about looking in the peripheral blood as well as the spleen. * do you have any other way of marking donor cells (eg a congenic pair or another dye) to rule out a problem with your donor cells. I haven't worked with donor PBMCs (just LNs or spleen) and I suppose it is theoretically possible the majority of your cells are activated are getting caught up in the lungs (or other peripheral sites) and hence you aren't finding them back in the spleen. I would have thought that at least some of your cells should return to the spleen though. * how are you gating your samples? We had one student "loose" all their CFSE+ cells because they gated them out in another channel (because they are very bright there can be a lot of spillover). The following reference might be a help if you can get hold of it. I haven't read it myself but have heard Chris Parish talk about technical issues surrounding the use of CFSE. Parish, C. and H. S. Warren (2000). Use of the Intracellular Fluorescent Dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to Monitor Lymphocyte Migration and Proliferation. Current Protocols in Immunology, Wiley and Sons. Good luck!. Adrian -- ______________________________________________ Adrian Smith (Research Officer) T CELL BIOLOGY GROUP Centenary Institute of Cancer Medicine & Cell Biology Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA. Ph: (02) 9565-6198 Fax: (02)-9565-6103
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