Hi Lisa, I did some Treg-sorting on a Vantage. I also used 70 µm Nozzle, but only 12 psi. We also choose the top 3-5% of CD4CD25-DP cells from spleen and other organs. Were the cells naiv, or activated before. With what CD4+ cells have they been cocultured? It is known, that for example high IL-2 levels can overcome suppression. Or the CD4 were already too activated to be stopped. Long time ago, I made the experience with thymocytes, that after "high speed" (35 psi) the cells look as viable as with "normal speed"(12 psi, 5000/ sec), but the proliferation reached only 60-70% and took much longer. So I don“t know, if Tregs are sensitive to high speed sorting, but I guess, that other reasons are responsible for your observations. Best regards Steffen PS: Maybe you can include an additional marker for Tregs (e.g. GITR, described in J.Exp.Med.Vol. 201, No. 2, 2005, 181187 and elsewhere). Am 20.07.2006 um 21:30 schrieb lixin86: > Hi, > > I Sorted Mouse CD4+CD25+ Regulatory T cells from total spleenocytes on > BD FACSAria. My > customer asked me to sort around 5% top CD25+ of total CD4+ T cells. > After sorting by 70 > nozzle on either 70 psi or 35 psi, the purity is around 98-99%. > However, the sorted > population had no suppression function. > > Can anyone tell me your kind advice? > > Lisa > -- > ******************************** > BC Research Institute, UBC > Room 212, 950 W 28th Ave. > Vancouver, BC V5Z 4H4 > Canada > Tel: 604-875-2000 ext 5987 > Fax: 604-875 3597 > =============================== Dr. Steffen Schmitt FACS und Array Core Facility NMFZ- Johannes Gutenberg Universität Obere Zahlbacherstr. 67 55131 Mainz Tel.: +49 6131 39 30219 Fax: +49 6131 230506 www.facslab.toxikologie.uni-mainz.de ==============================Received on Thu Jul 27 12:18:00 2006
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