guanine phosphoribosyl transferase (Hprt1). This measure is calculated with the following formula: 2Ct(Hprt1)-Ct(nos2) where Ct(Hprt1) and Ct(nos2) represent the cycle number where the signal is amplified to a threshold value that is held constant between experiments. nos2 transcript was amplified using the following primer sequences: ctcactgggacagcacagaa and gcttgtctctgggtcctctg (Integrated DNA Technologies). Proliferation assay FRC.5 cells were plated at the amounts indicated, where ‘% of FRC’ refers to the percentage of FRC.5 cells relative to the total T cells in culture, in wells of a 96 well culture-treated dish 6 to 8 hours prior to the addition of T cells. OTI-GFP, Th1, Th2 and OTI-IFNc2/2 T cells were purified as described above and labeled with either 2 mM CFSE or 4 mM eFluor670 proliferation dyes. 16105 T cells were added to FRC.5 cultures. Where indicated, T cells were activated with peptide-loaded BMDCs (16104 per well), plate-bound aCD3 (2 mg/ml), plate-bound aCD3 (2 mg/ml) and soluble aCD28 (2 mg/ml), or PMA (2 ng/ ml) and ionomycin (20 ng/ml). BMDCs were matured with 1 mg/ ml LPS 1 day before addition to cultures and pulsed with SL8 peptide (100 ng/ml) or Ovalbumin peptide 323–339 (250 ng/ml) for 1 hour. aCD3 was plated 16 to 24 hours prior to the addition of FRC.5 cells. Where indicated, T cell proliferation was augmented with the addition of various drugs, cytokines and antibodies at the start of the assay. Cultures were stained or fixed at the times noted and analyzed by flow cytometry. T-DC coupling assay For in vitro T-DC coupling, BMDCs were matured with LPS (1 mg/ml) 1 day before priming and pulsed with SL8 peptide (100 ng/ml) for 1 hour. BMDCs and na¨ıve OTI cells were labeled with 4 mM DDAO and 4 mM eFluor670, respectively. OTI cells were mixed with BMDCs at a ratio of T-DC 2:1. FRC.5 cells were added in the amounts indicated and the cell mixture was immediately centrifuged for 1 min at 228xg and incubated at 37uC. Cell mixtures were fixed at 15 minute intervals with an equal volume of warm 4% PFA in PBS. Cells were analyzed by flow cytometry for quantification of the percentage of couple formation, calculated as the number of T cells in the double- positive quadrant (eFluor670+ DDAO+) versus the total number of T cells (eFluor670+). Microscopy The following techniques were used to image calcium flux in OTI T cells. Wells of a Lab-Tek II chambered coverglass system (Nunc) were plated with 7.56104 FRC.5 cells or fibronectin (1 mg/ ml) 16 hours prior to the addition of BMDCs and OTI T cells. BMDCs were treated with LPS (1 mg/ml) 24 hours prior to the start of the assay and pulsed with SL8 peptide (100 ng/ml) for 1 hour. Na¨ıve OTI-GFP cells were selected as described above and labeled with 4 mM FURA-2 AM for 45 minutes at 37uC. 7.56104 peptide-loaded BMDCs were added to wells containing FRC.5 cells or fibronectin coating 5 minutes before the addition of 26105 labeled na¨ıve OTI cells. Interactions between BMDCs and OTI cells were visualized on a modified Zeiss Axiovert 200 M microscope with a 10x objective (Carl Zeiss). The microscope was fitted with dual excitation and emssion filter wheels, a Coolsnap-HQ camera (Photometrics) and a heated, motorized stage. Metamorph (Universal Imaging) was used as the imaging and control software. Data from DIC images, FURA emissions at 340 nm and 380 nm and green fluorescence were collected at 15- second intervals over a 20 to 30 minute period. Calcium flux was assessed based on the ratio of FURA-2 AM emissions at 340 nm to Regulation of T Cell Priming by Lymphoid Stroma those at 380 nm, with increased ratios indicating calcium flux. Microscopy of NOS2 levels in mixed cultures utilized NOS2- FITC conjugates as described for FACS, utilizing cultures in which DC and/or T cells were separately pre-labeled prior to the initiation of cultures with FRC. Data was collected on a wide-field Zeiss 200 M microscope and analyzed in Metamorph. The following techniques were used to image T cell clusters. 16104 FRC.5 cells or fibronectin were plated as described above. 6 hours later, 16106 na¨ıve OTI-GFP cells and 16105 SL8 loaded BMDCs were added in the presence or absence of 1400W. Prior to imaging, chambers were spun down at 400xg for 1 minute. Images were captured on a modified Nikon Eclipse TE2000-S (Nikon). The microscope utilized a CSU10 confocal scanner unit (Yokagawa) and a XR Mega S-30 camera (Stanford Photonics). A custom build of Micromanager was used to control image acquisition. Data from DIC images and green fluorescence were collected. Cluster area measurements were conducted using the FIJI software suite of ImageJ. Measurement of NO production Nitrite concentrations in culture supernatant were measured by the Griess assay as described previously[19]. Briefly, the culture supernatant was mixed sequentially with an equal volume of 1% sulfanilamide and 0.02% N-(1-naphthyl)ethylenediamine (0.5 M HCl) in a 96-well microplate. The absorbance at 540 nm was measured using a microplate reader, and the nitrite concentrations were derived from standard curves. Assessment of cell cycle status Propidium iodide (PI) and Ki67 stains were performed on the proliferation assays described above. Cultures were spun down at 400xg, medium was aspirated and replaced with 70% ethanol for PI labeling or PBS containing 5% FCS and 0.5% saponin (Sigma- Aldrich) for Ki67 staining. Cultures were incubated for 1 hour in 70% ethanol prior to washing and subsequent PI staining (10 ug/ ml in PBS containing 5% FCS). Percentage of cells actively cycling (in S, G2, or M phase) was calculated by taking the number of cells past the initial G0/G1 labeled peak and dividing by all live cells. Ki67 staining was performed immediately upon the addition of saponin medium. Cells were washed with PBS containing 10% FCS and analyzed by flow cytometry. Quantification of endogenous OVA-specific CD8 cells Where indicated, mice were immunized with either BMDCs loaded with peptide or aDEC205-OVA conjugates. BMDCs were loaded with SL8 peptide (100 ng/ml) for 60 to 120 minutes at 37uC. Mice were immunized with a subcutaneous injection of 7.56105 BMDCs per flank in the presence of 200 ng/ml LPS in PBS. DEC205-OVA conjugates (3 mg) were administered subcu- taneously in both flanks. At the times indicated, mice were euthanized and peripheral lymph nodes were excised. Lymph node cells were stained with R-PE conjugated MHC-I pentamer specific for the OVA peptide SIINFEKL in PBS containing 2% FCS. Subsequently, cells were stained for CD8 and CD19 and analyzed by flow cytometry. Acknowledgments We thank S. Peck and J. Wong for assistance in maintenance of microscopes and S. Jiang for expert technical assistance with cell sorting. We thank Sebastian Amigorena for support during establishment of the stromal lines. We thank C. Crane, E. Thornton, P. Beemiller, and J. Engelhardt for technical assistance and C. Sorensen for critical review of this manuscript. PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e26138