Bovine eyes (50 eyes for the entire study) were obtained from a
local abattoir (SCAN West, Skara, Sweden) and lenses removed
within 4 hours after death under aseptic conditions. The entire eye was
dipped in 70% ethanol for 30 seconds before it was opened from the
posterior. The dissection procedure was performed with sterile
instruments under a laminar flow hood. After removal of the vitreous,
the zonula fibers were cut, and the lens was lifted to a sterile filter
paper where it was gently rolled to remove any adhering vitreous or
ciliary body pigment. The lens was then pinned to a paraffin plate, the
capsule epithelium cut along the equator, and the whole lens placed
upside down in a 35-mm2 culture dish (Techno
Plastic Products, Trasadingen, Switzerland). The edges of the lens
capsule were pressed down to the plastic surface by fine forceps and
the bulk of the lens lifted from the dish, leaving the pieces of
capsule epithelium adhering to the plastic, cell side up. Some drops of
culture medium were applied to the epithelium specimens to prevent
drying, and the dishes were put in a humidified
CO2 incubator (5% CO2,
37°C) for approximately 2 hours to allow firm attachment of the
capsules. Another 1 ml of culture medium was then added, and the
capsules were left for 2 days before the first trypsinization.
Typically, BLECs from 10 explants were pooled and subcultured in 25- or
80-cm2 culture flasks (Nunclon; Nalge Nunc,
Napierville, IL) by trypsin-EDTA treatment (0.25%, from Sigma, St.
Louis, MO). The medium, which was changed twice a week, was M199 with
10% fetal calf serum, supplemented with 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin (all from Sigma). For most experiments, cells were seeded
in 96-well culture plates suitable for fluorescence assays (Corning
Costar, Cambridge, MA) at a density of approximately
105 cells/well and left overnight to yield a
confluent monolayer. For annexin labeling, cells were seeded on 13-mm
round coverslips (BDH, Poole, UK) in 24-well culture dishes (TPP).
Cells used in experiments were generally from the third to eight
passage. Cell morphology did not change between those passages, and no
increase or decrease of proteasome or caspase-3 activity in untreated
cells was seen with passaging.