Control and diseased corneal samples were thawed on ice, placed in 400 μL of lysis buffer (Bio-Rad cell lysis kit; Bio-Rad, Hercules, CA, USA) and sonicated 10 times for 10 seconds. Lysates were incubated at 4°C under gentle agitation for 2 hours. Protein concentration was measured by BCA assay (Thermo Scientific Pierce, Rockford, IL, USA). Lysates with a concentration between the range of 0.1 to 1.1 mg/mL were selected (n = 21; PKP, n = 17; DALK, n = 4) and further processed using the Bio-Plex Pro Human MMP Panel, 9-Plex (Bio-Rad) following the manufacturer's instruction. Briefly, lysates were incubated with specific anti-MMP antibodies, each type of which was covalently linked to uniquely colored, fluorescently labeled magnetic beads. Beads were washed three times to remove unbound proteins, incubated with a specific anti-MMP biotinylated detection antibody, washed again, and incubated with a streptavidin-phycoerythrin conjugate serving as a reporter. Fluorescent signals from both the beads and phycoerythrin were measured in the multiplex suspension using MAGPIX technology and compared to standards of known concentrations. Lower limits of detection for MMP1, 2, 3, 7, 8, 9, 10, 12, and 13 were 35, 450, 116, 5.4, 1.5, 24, 1.6, 1, and 4.9 pg/mL, respectively. Standard curves were run in duplicate, and all quality control reagents were within range. Minimal disparity was measured between the duplicates that all passed the manufacturer's internal quality control test before being processed by the calculation algorithm. Results were analyzed using Bio-Plex Data Analysis software (Bio-Rad) and the concentration of each individual MMP per corneal sample was calculated and normalized per milligram of total protein.