Cell transfers in vivo.
1| Harvest splenocytes from 5 to 8 week old mice.
2| Osmotically-lyse the erythrocytes and incubate the single cell suspensions with FITC-conjugated anti-CD4 and biotin-conjugated anti-CD45RB followed by incubation with anti-FITC microbeads.
3| Purify CD4+ T cells by magnetic isolation using the Auto MACS sorter (Miltenyi Biotec). For isolation of CD4+CD45RB high naive cells, after releasing the beads, incubate the purified CD4+ T cell suspension with α-biotin microbeads followed by separation using the Auto MACS. In all of our experiments, 99% of these cells were positive for CD4 and high for CD45RB.
4| For the first intra peritoneal (i.p.) injection, inject Thy1.2+ C.B-17 scid mice with 4 × 10⁵ fresh Thy1.1+CD25– CD45RBhighCD4+ congenic cells.
5| After 6 or 7 days, administer an i.p. injection of PBS or 0.8-1 × 10⁶ freshly isolated Thy1.2+ Treg cells. Control mice receive PBS in both the injections. C57BL/6 Rag1–/– or Rag2–/– mice (CD45.1) can be used as recipients C57BL/6 donor cells were transferred. After 6 or 7 days, they received an i.p. injection of PBS or 1 × 10 6 freshly isolated CD45.1+ Treg cells. Cells were transferred in 100ul of PBS/ mouse. On day 6-10 after Treg cells were transferred, mesenteric lymph node cells or lamina propria cells were isolated (using the protocol from Current protocols in Immunology) and Annexin V and Propidium iodide staining was performed to measure apoptosis using flow cytometry. TUNEL staining was also done on cryo-sections of the gut to measure apoptosis of the effector cells in the lamina propria of the gut followed by confocal immunofluorescence analyses.