Begin by sterilizing polytetrafluoroethylene, or PTFE-coated slides by immersing them in 70%ethanol for 10 minutes in a biosafety cabinet. Allow the slides to air-dry in a sterile 100-millimeter cell culture dish. Place each slide in 1 new 100-millimeter cell culture dish and add 200 microliters of serum-containing cell culture medium into each rectangle.
Then place a handheld 254-nanometer UV light on the 100-millimeter cell culture dish with the bulb directly facing the slide for 20 minutes. Thaw the frozen aliquots of the outer segments, or OS, in a 37-degree Celsius water bath. Then spin the OS at 2, 400G for five minutes at room temperature.
Immediately aspirate the supernatant in the biosafety cabinet using a pipette and resuspend the OS in PBS. Now, aspirate the medium from the slides and place up to 500 microliters of the 2 x 10 to the 8th OS per milliliter PBS solution of the 2 x 10 to the 8th OS per milliliter PBS solution on each slide rectangle. Place a handheld 254-nanometer UV light over the cell culture dish with the bulb directly facing the OS for 40 minutes.
Collect OS-PBS solution in a 1.5-milliliter tube. Wash the rectangle of the coated slide with 200 to 500 microliter of PBS and collect each wash in the microcentrifuge tube. Then spin down the OS-PBS solution at 2, 400G for five minutes at room temperature.
Aspirate the PBS in the biosafety cabinet with a pipetter and resuspend the pellet in 500 microliters of standard medium to be used for the retinal pigment epithelium, or RPE cultures. Place 10 microliters of the suspension in a new microcentrifuge tube. Dilute 50 to 100 times with more cell culture medium, and count the OS with a hemocytometer.
Dilute the photo-oxidized OS, or OxOS suspension, at a 5.6 x 10 to the 7th OS per milliliter concentration into a standard RPE medium. Add phagocytosis bridging ligands, which facilitate OS uptake and lipofuscin accumulation. Then aliquot OxOS into single-use stocks and snap-freeze in liquid nitrogen.
Thaw OxOS aliquots by diluting the frozen aliquot in 45 microliters of cell culture media before adding it to RPE transwells to induce lipofuscin accumulation. Then characterize the oxidized OS by measuring the OxOS emission spectrum using Lambda scanning on a confocal microscope. Confocal imaging showed that treated OS had increased autofluorescence compared to untreated OS.
