Inflammatory and Barrier Marker Expression

The expression of inflammation markers, like NF-κB, TNF-α, IL-1β and NLRP3, was analyzed. The mRNA expression of tight junction’s marker occludin and the glycocalyx marker galectin-3 was also investigated in the porcine model (Table 2). Briefly, after incubation, the corneas were cut into small pieces and incubated in 800 µL Lysis Buffer (Miltenyi Biotec, Köln, Germany) for 1 h at 37 °C on a shaker. The mRNA was isolated from porcine cornea explants and reverse transcribed using the MultiMACS mRNA and cDNA synthesis was done with cDNA Synthesis Kit on the MultiMACS™ M96 Separator (Miltenyi Biotec, Köln, Germany) according to the manufacturer’s protocol. After cDNA synthesis, a quantitative real-time PCR (qRT-PCR) was performed using the SsoAdvanced Universal SYBR^® Green Supermix (Bio-Rad, Feldkirchen, Germany) in a thermal cycler (Bio-Rad CFX96™ Real-Time System, Bio-Rad, Feldkirchen, Germany), as described previously [56 (link)]. β-actin (ACTB) and Ribosomal protein L 4 (RPL4) were used as housekeeping genes.