Induction of apoptosis in human corneal epithelial cells in vitro.
OBJECTIVE
The purpose of this study was to evaluate exhausted-medium-induced apoptosis in human corneal epithelial (HCE) cells in vitro.


METHODS
Confluent HCE cells were maintained in tissue-culture medium for 5-7days. The supernatant was harvested and fresh HCE cells were exposed to the exhausted supernatant for 24-48 h over a range of pH conditions. The exhausted medium was assayed for cytokines interleukin 6 (IL-6), IL-8, interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) by ELISA. Apoptotic cells were assessed using Hoechst 33342 Propidium iodide and AnnexinV-FITC staining and by the TUNEL assay.


RESULTS
Human corneal epithelial (HCE) cells exposed to exhausted medium demonstrated a high incidence of apoptosis, which increased over time to 87+/-5% after 48 h. Apoptosis was independent of pH. Cytokine levels were not elevated in the exhausted medium.


CONCLUSIONS
This study suggests that exhausted medium can be used as a simple, rapid and potent positive control for apoptosis of HCE cells.
Kim
J M
JM
Cooperative Research Centre for Eye Research and Technology, University of New South Wales, Sydney, Australia.
Stapleton
F
F
Willcox
M D
MD
D017304:Annexin A5; D017209:Apoptosis; D001562:Benzimidazoles; D002461:Cell Line, Transformed; D004396:Coloring Agents; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D016207:Cytokines / Q000032:analysis; D053938:DNA Fragmentation; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D020287:In Situ Nick-End Labeling; D011419:Propidium
D017304:Annexin A5; D001562:Benzimidazoles; D004396:Coloring Agents; D003470:Culture Media; D016207:Cytokines; D005456:Fluorescent Dyes; D011419:Propidium; D016650:Fluorescein-5-isothiocyanate; C017807:bisbenzimide ethoxide trihydrochloride