Fresh splenocytes

were prepared as described before 8. Cultured adherent cells were detached with disassociation buffer Fulvestrant chemical structure (GIBCO). Cells were stained with Abs (Supporting Information Table 2) at 4°C for 30 min. For two-step staining, biotinylated Ab were subsequently detected using streptavidin conjugates (Supporting Information Table 3). 1 μL of DAPI (0.25 μg/mL) and 20 000 of non-fluorescent particles (counting beads) (Spherotech) were added into each sample and the sample acquired using a CyAn flow cytometer. Data were analyzed by FlowJo software (TreeStar). For cell sorting, cells were prepared and stained as above. Cells were purified by MoFlo cell sorter (Dako Cytomation). Frozen IL-7−/− spleens kindly donated by Daniella Finke (University of Basel, Switzerland) were sectioned and prepared as described before 22. Cells were stained with the Abs (Supporting Information Tables 2 and 3). Anti-CD4 mAb was directly conjugated using the Alexa Fluor 647 mAb labeling kit (Invitrogen) according to the manufacturer’s instructions. Biotinylated Ab buy FK506 were detected by streptavidin Alexa fluor 555 (Invitrogen). FITC-conjugated Ab were detected using rabbit anti-FITC Ab (Sigma), then goat anti-rabbit FITC Ab (Southern Biotech). Confocal images

were acquired using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss) and analyzed using LSM510 software. Cell suspension were made from spleens of Rag−/−γc−/− and CD3εtg mice as described previously 6. Briefly, CD4+ cells were enriched from CD11c+-depleted populations using MACS anti-mouse CD4 microbeads (Miltenyi Biotech) according to the manufacturer’s protocol. Enriched CD4+ splenocytes contained between 8 and 30% LTi-like cells 4. Briefly, 2–5×104 of CD4+-enriched cells were cultured and incubated for 4 days prior to further FACS analysis. SSCL and white pulp stromal cells were obtained as described previously

23. CD4+ enriched populations were cultured Methamphetamine with DMEM with 10% FCS supplemented with 1% penicillin/streptomycin and 2 mM L-glutamine on irradiated (2000 Rad) CD45−podoplanin+ SSCL with or without 2 μg/mL blocking anti-IL-7 Ab (R & D). Controls were cultured in the absence of any stromal cells with or without 0.01 g/mL recombinant IL-7 (Pepro Tech). All cultures were incubated for 4 days. The recovered cell number calculated as: viable LTi-like cell number is equal to the DAPI−CD3−CD11c−B2220−CD4+ cell number shown in FACS plot divided by the bead number shown in FACS plot then times 20 000. CD45−podoplanin+ SSCL were frozen in liquid nitrogen, and high-purity cDNA was obtained from purified mRNA, using μMacs One-step cDNA synthesis kit, according to the manufacturer’s instructions (Miltenyi Biotech). β-Actin was used as the housekeeping gene for sample normalization, prior to amplifying the target genes of interest. RT-PCR was performed using SYBR Green with primers (Supporting Information Table 4).