To begin, calculate the number of wells needed for the experiment. Then, thaw an appropriate amount of regular OS sample. Add enough RPE media to achieve a final OS concentration of four times 10 to the sixth per milliliter.
Remove the apical medium and add 50 microliters of four times 10 to the sixth OS per milliliter with appropriate concentrations of bridging ligands. After OS addition, incubate the samples for various time points. At the end of the incubation, add 16.67 microliters of four times Laemmli sample buffer with protease inhibitors to lyse both the cells and the overlying OS containing supernatant.
Using a P200 pipette, scratch the Transwell surface and collect the combined cell supernatant and cell lysate together. Vortex and leave at room temperature for 30 minutes for thorough denaturation. Western blot of the rhodopsin showed that the intact rhodopsin band was indistinguishable in control and UAM laden RPE cells.
However, the cleavage products of rhodopsin were higher in the UAM group, suggesting some mild degradative dysfunction in the phagolysosomal system. The equal levels of GAPDH indicate that the cell count between wells is equal. Different rhodopsin antibodies recognize different degradation fragments.
4D2 recognizes the end terminus of rhodopsin which is intact until the last steps of lysosomal degradation. In contrast, 1D4 recognizes the C terminus of rhodopsin which is degraded earlier in the phagolysosomal process.
