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N. G. Lee, N. Varshney, M. Lin, A. A. Fawzi, D. Hinton, T. Lee; Mechanical and Chemical Disruption of Cell Adhesions of Retinoblastoma Y79 Cells Causes Increased Apoptosis and Decreased Proliferation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1698.
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Retinoblastoma (Rb) cells form aggregates both in vivo and in vitro. Attachment of normal cells to a suitable substrate is necessary to initiate cell proliferation. It has been shown that Rb cells adhere to each other via filopodia and villi. We hypothesized that disruption of the cell-cell adhesions would negatively affect Rb cell proliferation. We have conducted an experiment of in vitro Rb cells (Y79) to study the effect of dissociating the cell-cell bonds on cell survival and apoptosis using either chemical (Accutase) or mechanical disruption of adhesions.
Y79 cells were divided into 3 experimental conditions: Control, Accutase, and mechanical disruption. Control cells were gently resuspended in medium. The "Accutase" cells were resuspended in 200 ul of Accutase for 10 minutes. The last group of cells underwent vigorous pipetting. At time points 0, 24, and 48 hours, trypan blue exclusion and flow cytometry (FC) was performed on the 3 cell groups. FC was performed with Annexin-V FITC and Propidium Iodide for assessment of apoptosis and cell survival. A positive control for flow cytometry was established with Y79 cells that were suspended in Etoposide. Scanning electron microscopy was performed to study the structure of the adhesions between the cells following the various treatments.
Y79 cells underwent trypan blue exclusion after various time points following the three treatment conditions. Within 30 minutes of resuspending, the control cells had a viability of 93% compared to the mechanical (83%) and chemical disruption (86%). After 24 hours, there was no significant difference in viability of the cells across the experimental groups. There was a notable decline in the total number of cells at 30 minutes and 24 hours in the Accutase and pipetted groups as compared to the control group but no difference across groups at 48 hours. Compared to controls, FC showed an increase in apoptosis in both treatment groups at baseline and at 24 hours, but not at 48 hours. Microscopic examination showed that the Y79 cells had re-aggregated and were arranged in clumps at 24 and 48 hours.
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