Digital light fluorescence microscopy system

HLEC colonies were established from 4 different cadaveric human donor corneas. Cells from each cornea were seeded into two wells of a 6 well plate, yielding 8 cultures in total. Once macroscopically visible colonies had formed, the cells were incubated with 1ml of Qdot labeling solution (as previously described) for 60 minutes. The cultures were washed three times with culture medium then incubated as normal. At 24 hours post labeling, images were collected using the Olympus digital light/fluorescence microscopy system. Ten images were collected from different HLEC colonies in each of 8 replicate wells. The number of Qdot containing cells was counted and this was expressed as a percentage of the total number of cells. The cultures were then passaged and reseeded onto fresh feeder layers at a density of 2000 cells per well of a 6 well plate. Six days later (day 7 following the initial labeling of cells), the percentage of cells containing Qdots was again evaluated. The cells were then passaged for a third and final time and the percentage of cells containing Qdots was counted again 14 days after the initial labeling.

Cells were imaged using both fluorescence and confocal microscopy. An Olympus digital light/fluorescence microscopy system (Olympus, Japan) was used to capture fluorescence images. A HBO lamp with a 450 – 550nm band pass filter was used for excitation and a 650nm long pass filter for detection. These images were analysed using image analysis software (Soft Imaging System GmbH, Munster, Germany). Confocal images were captured using a Zeiss LSM 510 confocal microscope (Zeiss GmbH, Germany). A 548nm Argon laser was used for excitation and a 650nm long pass filter for detection.

HLEC were cultured in Permanox 4-well chamber slides and then labeled with 200μl of Qdot nanocrystal labeling solution. Slides containing cells that had been through the labeling process, with the exception of the Qdot reagent, were used as negative controls. The ratio of living to dead cells was assessed at 24 hours post labeling and again at 72 hours post labeling using a live/dead assay kit (LIVE/DEAD® Viability Cytotoxicity Assay Kit, Invitrogen/Molecular Probes). Images were captured using the Olympus digital light/fluorescence microscopy system, and the proportion of live and dead cells was counted. This was performed three times, a different cadaveric human donor cornea being used each time (n=3).