Paul, We've done a lot of these MLRs in vivo and in vitro using CFSE (e.g. J.I. 166:973, 2001). You definitely use the same labeling procedure whether you're going to put the cells in a mouse or in a dish. 48 hours is too early for 4/5 divisions in the in vitro (or in vivo) MLCs. You're most likely picking up stimulators, especially if they are autofluorescent due to irradiation or mitomycin C treatment. Do you see this population in your autologous controls, too? How are you identifying the donor/responder cells in these experiments? Identifying them by CFSE is not enough - you need some other marker (i.e. Thy1.1 vs. Thy1.2, Ly5.1 vs. Ly5.2, or donor-specific MHC I) to distinguish your donor/responder T cells from your recipient/stimulator cells, both in vivo and in vitro. Thy1.1 B6 mice are available from Jackson (normal B6's are Thy1.2). Ly5 congenics are also commercially available. Using this type of congenic system will solve a lot of your problems. Also, the in vitro MLRs give much more variation from experiment to experiment (talk to anyone who does them using H3-TdR, they'll say the same thing), and they're trickier to get working well. The in vivo MLRs are extremely consistent. Hope this helps, Andrew -- Andrew D. Wells, Ph.D. Assistant Professor Department of Pathology and Laboratory Medicine University of Pennsylvania Joseph Stokes, Jr. Research Institute Biesecker Liver Disease Center The Children's Hospital of Philadelphia phone: (215) 590-8710 fax: (215) 590-7384 mailing address: 802 Abramson Research Center 3516 Civic Center Boulevard Philadelphia, PA 19104
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