performed experiments and analyzed data; M.C.Z. to the PC lineage. In this study, we investigated the functional role of CD9 on GC-B cells. Tonsillar tissue section staining revealed that CD9+ GC-B cells localized in the light zone FDC area. Consistent this, CD9+ GC-B cells survived better than CD9? GC-B cells in the presence of HK cells, an FDC Sodium formononetin-3′-sulfonate line, in a cellCcell contact-dependent manner. The frozen tonsillar tissue section binding assay showed that CD9+ GC-B cells bound to the GC area of tonsillar tissues significantly more than the CD9? GC-B cells did and that the binding was significantly inhibited by neutralizing anti-integrin 1 antibody. Furthermore, CD9+ cells bound to soluble VCAM-1 more than CD9? cells did, resulting in activation and stabilization of the active epitope of integrin 1. All together, our data suggest that CD9 on GC-B cells contributes to survival by strengthening their binding to FDC through the VLA4/VCAM-1 axis. location of CD9+ Sodium formononetin-3′-sulfonate GC-B cells in the FDC area prompted us to determine whether CD9 has a functional role in the interaction Sodium formononetin-3′-sulfonate between GC-B cells and FDC. To address this question frozen section Rabbit Polyclonal to EPS15 (phospho-Tyr849) binding assay was performed as described by Freedman et al. [29,30]. In short, GC-B cells were labeled with 1?M Calcein AM (Invitrogen) for 15?min at room temperature in PBS. Cells were washed twice with serum containing media and resuspended at 3??107?cells/mL. One hundred microliters of the cell suspension were placed onto frozen sections of tonsil and incubated at 37?C for 30?min. After incubation, slides were fixed in 3% glutaraldehyde in PBS overnight at 4?C. Slides were washed, mounted with anti-fade mounting medium containing DAPI (Invitrogen), and then examined by fluorescence microscopy. For blocking experiments, cells were preincubated with neutralizing anti-integrin 1 (R & D Systems) or isotype control antibodies (10?g/mL) for 30?min before applying to tissue sections. 4.6. sVCAM-1 binding and detection of an activated form of CD29 L3055 cell subclones L3055-12 and L3055-33 were maintained as described previously . L3055-12 and L3055-33 (5??105?cells per 100?L of the complete media) were incubated with soluble VCAM-1-His Tag (10?g/mL, R & D systems) at 37?C for 30?min. Binding of soluble VCAM-1 was detected by incubating the cells with the FITC-conjugated anti-His antibody for 20?min at 4?C. As a negative control, the same amount of soluble 41BB-His Tag (R & D systems) was added. In some experiments, soluble VCAM-1 was preincubated with neutralizing anti-VCAM-1 antibody (10?g/mL, Beckman Coulter, Indianapolis, IN) before incubation with the cells. To detect an activated form of CD29, the cells were incubated with soluble VCAM-1, followed by staining with PE-conjugated anti-CD29 (clone, HUTS21). 4.7. Statistical analysis Statistical analysis and graphic presentation were carried out with GraphPad Prism 4.0 (GraphPad, La Jolla, CA). Results are presented as means of triplicate assays plus SEM. The statistical significance of differences was determined by Students em t /em -test; em P /em ? ?0.05 was considered significant. Author contributions S.O.Y. planned experiments, performed experiments, analyzed data, and wrote the paper; I.Y.L. performed experiments and analyzed data; X.Z. performed experiments and analyzed data; M.C.Z. performed experiments; Y.S.C. planned experiments and contributed reagents. Conflict of interest No potential conflicts of interest were disclosed. Acknowledgements This work was supported in part by NIH grants R01CA121039 to Y.S.C. and P20GM103501 to S.O.Y. Appendix A.?Supplementary data Supplementary data 1: This document file contains Supplementary Figs. 1C4. Click here to view.(457K, pdf).