Cy5.5 (104), CD44-APC (IM7), CD62L-PE (MEL-14) CD69- biotinylated (H1.2F3), CD138-PE (281-2), CXCR5-biotinylated (2G8), Fas-biotinylated (Jo2), GL7-FITC (GL7), PD-1-PE (J43) and streptavidin-APC were from PharMingen (BD Bioscience, Oxford, UK) or eBioscience (Hatfield, UK). Populations or OTII single cells were sorted by flow cytometry (MoFlo, DakoCytomation, UK) and the purity was routinely .90%. Final analysis and graphical output were performed using FlowJo software (Treestar, Costa Mesa, CA). Ex vivo restimulation and intracellular cytokine and immunoglobulin staining Various days after primary or secondary immunization popliteal or brachial LN cells were incubated at 106106 cells/ml with 10 mM free 323–339 OVA-peptide for 5 h prior to cytokine detection. Intracellular FACS staining was performed using Cytofix/cytoperm kit (Becton Dickinson, Oxford, UK) according to manufacturer’s instructions. Anti- IL-4-APC (11B11) is from PharMingen. Intracellular staining to detect antibody-producing plasma cells was performed using BD cytofix/cytoperm kit. Goat anti-IgG1- alexa633 and anti-IgG2a-FITC were from Molecular Probe (Invitrogen, Paisley UK) and Southern Biotech (Cambridge BioSscience, Cambridge, UK), respectively. Foxp3, GATA-3 and Helios detection by intracellular flow cytometry Intracellular staining was performed with the Foxp3 staining buffer kit, according to the manufacturer’s protocol (eBioscience, Hatfield, UK). Anti-Foxp3-e-Fluor 450 (FJK-16s), anti-GATA-3- e-Fluor 660 (TWAJ), anti-Helios-Pacific Blue (22F6) and anti- Helios-PE (22F6) [27] were from BioLegend (Cambridge, UK) or eBioscience (Hatfield, UK). Immunohistochemical analysis For in situ study of immune responses 5 mm cryostat sections were taken from snap-frozen LN for immunohistology as described in [60]. After cutting the first sections, which were used for immunohistology, two 25 mm sections of LN were cut, placed in a polypropylene microfuge tube, and stored at 270uC. Sections were fixed in acetone at 4uC for 20 minutes and air dried. The staining to reveal IgG1, IgG2a have been described in [57,58,70]. Real-time RT-PCR Real-time semi-quantitative RT-PCR on population, or LN sections, was performed as previously described [5,71]. Briefly, probes and primers were designed using Primer Express software (Applied Biosystems) and sequences are detailed in Table S1. When possible, duplex were performed with b2-microglobulin and a target gene. Probes for cytokines and transcription factors were detected via a 59 label with FAM, while probe for b2- microglobulin was 59 labeled with NED. Single cells were collected in 384 well plates containing 2 ml of cell-to-signal lysis buffer (Ambion, UK). mRNA were specifically reverse-transcribed and the PCR performed by adding 5 ml of a mix containing the Multiplex enzyme RT-PCR master mix (Qiagen Multiplex Probe RT-PCR kit), primers and probes. Details of the strategy and efficiency of the duplex and triplex RT- PCR on single cells are shown in the Figure S1. Standard reaction conditions for the TaqMan RT-PCR or PCR were used on the ABI 7900. Helios Marks In Vivo Differentiated CD4 Th2 Cells In vitro Th2 polarization Total LN OTII cells were incubated at 56106 cells/ml in 6 well plates with 1 mM free 323–339 OVA-peptide (Alta Bioscience, University of Birmingham, UK) in complete medium, plus IL-4 (10 ng/ml) and neutralizing anti-IL-12 (C17.8) (5 mg/ml) plus anti-IFN-c (X.MG1.2) (5 mg/ml) for 6 days. Restimulation was performed with anti-CD3 (145-2C11) coated (5 mg/ml) plus soluble anti-CD28 (37.51) (1 mg/ml) for 5 h at 37uC. Cytokines were from PeproTech and antibodies from Insight Biotech. Statistics Statistical analysis was performed using a two-tailed non- parametric Mann-Whitney test. Values of p,0.05 were consid- ered significant and all the p-values are indicated on the figures. Supporting Information Figure S1 Technique and strategy for duplex and triplex RT-PCR at the single cell level and its validation. Consecutive two-fold dilutions of the mRNA template were amplified individually for IL-4 and each transcription factor gene product. The RT-PCR for GATA-3 or Helios were set up to work in triplex with b2microglobulin and IL-4 primers and probes, while Ikaros, c-Maf or NF-kB1 were set up to work with primers and probes specific for b2microglobulin only. A) CT plotted versus the 2 fold successive dilutions of mRNA, showing a linear quantitative relationship between the amount of mRNA template and the CT. This shows that RT-PCR run as simplex (open diamonds) or triplex (close diamonds) give equivalent results. Equations describing the trend lines are shown in each graph. We paid particular attention on the slope for each trend line and only accepted a difference of 0.1 between simplex and triplex RT-PCR. The correlation coefficients are also shown and are all close to 1. Pictures show the intensity of fluorescence as a function of the numbers of PCR cycles using SDS software (AppliedBiosystems) for varying numbers of FACS-sorted cells (32, 16, 8, 4, 2 see brown or purple lines) and 5 different single cells (see blue or green lines). Relative quantification for gene expression in single cells was achieved by setting thresholds (horizontal red line) within the logarithmic phase of the PCR and determining the cycle number at which the fluorescence signal reached the threshold (Ct). This shows that, as expected, the CT increases as the number of cells decreases. There is one blue flat line on each picture that shows a well without template that is with no FACS-sorted cell. B) Similar as in (A) for primer/probes that were run in simplex (open diamonds) or duplex (grey diamonds) RT-PCR. (PPT) Table S1 Sequences for the primers and probes used in this study as indicated in the materials and methods in the section real-time semi-quantitative RT-PCR. Se- quences are written from the 59 to the 39 terminus. a Assay-on- demand (Applied Biosystem). GT: Germline Transcripts. (DOC) Acknowledgments We are very grateful for the help and support provided by The Biomedical Services Unit at The University of Birmingham. Author Contributions Conceived and designed the experiments: KS ICMM AFC EM. Performed the experiments: KS EM SB. Analyzed the data: KS ICMM EM. Contributed reagents/materials/analysis tools: CB GD K-MT RB SC PK. Wrote the paper: KS ICMM EM. PLoS ONE | www.plosone.org 11 June 2011 | Volume 6 | Issue 6 | e20731