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D. M. de Bruin, M. Broekgaarden, T. Gorgels, T. G. van Leeuwen, D. J. Faber; Measuring Cell Death in Human RPE Cells Using Optical Coherence Tomography. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5551.
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© ARVO (1962-2015); The Authors (2016-present)
The disappearance of retinal pigment epithelial (RPE) cells is a key event in the development of late age related macular degeneration (AMD) and is thought to occur through apoptosis. This is accompanied by morphological changes that may be detected by optical coherence tomography (OCT). We used OCT to measure cell death in pelleted RPE cells using the attenuation coefficient (µt) of the signal. Control experiments were performed using flow cytometry (FACS).
Cells: The used RPE cells (ARPE19) were grown in medium (DMEM/ F12 1:1) under CO2 conditions. After a 6 weeks maturation period, the cells were ready for experiments. Cell death was induced by either a 30 minute shock or by constant exposure to cell death inducers. Samples of 5*106 cells were taken for FACS analysis. The remainder of the cells was used for OCT imagingOCT: A dynamic focusing TD-OCT system was employed in this study. The signal to noise ratio was 115 dB and the moving reference arm mirror allowed scanning speed of 1 depth profile per second with 4096 points per a-line. The µt was fitted using Beers law.FACS: FACS analysis was done by staining the cells using the Vybrant® Apoptosis Detecion kit #2. Approximately 5x106 cells harvested were stained using AlexaFluor® 488-Annexin V for apoptosis detection. PI was added to stain necrotic cells. Data analysis was performed using FlowJo v8.8.
Control experiments were performed on healthy cells only exposed to medium. Both FACS and OCT show that no increase in cell death was measured. OCT data reported a slight increase in µt during 6 hour monitoring with an average µt of 5.69±0.38. OCT measurements of a necrotic cells pellet induced by heat shock revealed a constant µt with an average of 3.87±0.39. OCT measurements of an apoptotic cell pellet induced by a shock of 10% EtOH caused a distinct change in µt; an initial increase (µt = 6.5 ± 0.4 at highest point at 3 hours), followed by a gradual decrease. All measurements were in agreement with FACS data.
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