RESUMO
Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment; hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.
