Protocol for the preclinical evaluation of gut barrier function and immune interaction in an HT-29/PBMC co-culture model

Here, we present a protocol for the pre-clinical evaluation of gut barrier function and immune interaction in an HT-29/PBMC co-culture model. We describe steps for culturing HT-29 cells and peripheral blood mononuclear cells (PBMCs), assembling the co-culture model, and testing cell viability. We then detail procedures for performing efficacy tests through stress challenges and barrier permeability assays. For complete details on the use and execution of this protocol, please refer to Abot et al. 1 • Steps to culture HT-29 cells to 70%–80% confluence before co-culturing • Isolate and prepare PBMCs for co-culture in RPMI 1640 medium with 10% FBS • Assemble co-culture by placing HT-29 cells on a Transwell insert above PBMCs • Evaluate cell viability and gut barrier function via cytotoxicity and permeability assays Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for the pre-clinical evaluation of gut barrier function and immune interaction in an HT-29/PBMC co-culture model. We describe steps for culturing HT-29 cells and peripheral blood mononuclear cells (PBMCs), assembling the co-culture model, and testing cell viability. We then detail procedures for performing efficacy tests through stress challenges and barrier permeability assays.