Tissue engineering has emerged as a strategy to combat the donor shortage of human corneas for transplantation. Synthetic corneal substitutes are currently unable to support the normal phenotype of human cells and so decellularized animal corneas have been deployed to more closely provide the topographical and biochemical cues to promote cell attachment and function. Although full thickness decellularized corneas can support corneal cells, the cells are slow to populate the scaffold and density declines from the surface. To avoid these problems, this protocol describes the stacking of alternate layers of decellularized porcine corneal sheets and cell-laden collagen hydrogel to produce a corneal construct. The sheets are obtained by cryosectioning porcine corneas, decellularizing them with

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear