Cells were freeze-thawed and harvested selleck chem onto Unifilter plates (Perkin-Elmer, Boston, MA, USA) and incorporation of 3H-thymidine was measured as counts per minute (cpm) using a liquid scintillation counter (Top-Count, Perkin-Elmer). Statistical analysis The outcome data from this study were not normally distributed. Therefore, differences among experimental groups were tested using Kruskal-Wallis analysis and medians were compared using Dunn��s test. In those instances where repeated measures were made on the same animals over time, the data for each animal were summed over the duration of the study and then Kruskal-Wallis analysis and Dunn��s test were performed on the sums. Differences were considered significant if P<0.05. All statistical analyses and graphing were formed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA).

Abbreviations BCG: Bacillus calmette-gu��rin; BALs: Broncheoalveolar lavage; CMI: Cell-mediated immune response; Cpm: Counts per minute; DCs: Dendritic cells; Tris: Di; PNPP: P-nitrophenyl phosphate; ELISA: Enzyme-linked immunosorbent assays; GALT: Gut-associated lymphoid tissues; H: Hour; IL: Interleukin; IFA: Incomplete freund��s adjuvant; i.p.: Intraperitoneal; IFLs: Isolated lymphoid follicles; MW: Molecular weight; M. bovis: Mycobacterium bovis; OVA: Ovalbumin; OD: Optical density; PFU: Plaque forming units; PPD: Purified protein derivative; RT: Room temperature; StDev: Standard deviation; TBST: Tris-buffered saline with tween 20; VIDO: Vaccine & infectious disease organization. Competing interests The authors declare that they have no competing interests.

Authors’ contributions HLW conceived of and designed the experiments and wrote the manuscript. RMB and SM performed the laboratory experiments. All authors read and approved the final manuscript. Acknowledgments We gratefully acknowledge financial support from the Agriculture Development Fund awarded to HLW and the excellent expertise offered by members of the Animal Care Group at VIDO. HLW is an adjunct professor in the Department of Biochemistry at the University of Saskatchewan. We gratefully acknowledge Dr. Hugh Townsend for his guidance with the statistical analysis. This manuscript is published with the permission of the Director of VIDO as journal series no. 651.

continuous renewal of the adult small intestinal epithelium is driven by intestinal epithelial stem cells (ISCs), which are located at or near the base of the crypt (2, 46, 54). Current evidence suggests that these cells self-renew Carfilzomib and give rise to daughter cells, termed progenitors or transit-amplifying cells, which actively divide and then differentiate into four terminally differentiated intestinal epithelial cell (IEC) lineages. Until recently, it was difficult to directly evaluate or isolate ISCs because of lack of valid biomarkers.