Dependence of cellular loss or entry of fluorescein on cellular activity.

<p>The proportion of hyperfluorescent L929 cells is shown after control (n = 12 wells) or MPS (n = 8 wells) treatment, for cells treated at 37°C with fluorescein, and then washed and observed at either 37°C or 4°C. Cultures held at 4°C do not release fluorescein (A). Conversely cells treated with fluorescein at either 37°C or 4°C, prior to observation at 4°C (the latter to prevent further change in fluorescein hyperfluoresence after treatment) are shown; cultures treated at 4°C appear to accumulate much lower levels of fluorescein (n = 4 wells) (B). Data shown are representative of several experiments; bars represent standard deviation and standard error in A and B, respectively.</p

Fluorescein and propidium iodine staining of hCTEpi cells treated with MPS.

<p>hTCEpi cultures were treated with hCTEpi growth medium containing 25% ReNu MultiPlus® MPS or PBS (control) and stained with Hoescht 33342, propidium iodide and fluorescein following an overnight incubation. Typical appearance of control cells is shown after Hoescht 33342 staining (A), propidium iodide staining (B) and fluorescein staining (C), with the equivalents for MPS-treated cells shown in (D) to (F) (in all cases n = 6 wells). The numbers of fluorescein hyperfluorescent cells clearly increased after MPS-treatment. Importantly hyperfluorecent cells were not also found to stain strongly with PI (one example is indicated by the circle). Data shown are representative of several experiments. Scale bars in (A) to (F) represent 100 µm.</p

Fluorescein staining of control cell populations.

<p>L929 cell cultures were treated with fluorescein and Hoescht 33342 prior to observation. Nuclear staining is shown in (A) and fluorescein staining in (B) with typical hyperfluorecent cells visible. Images were obtained using the ArrayScan®II system and cells categorized by fluorescein intensity with hyperfluorescent cells identified shown in (C). ‘Solution induced corneal staining’, as seen on a slit lamp biomicroscope, is shown (D) for comparison, in which characteristic hyperfluorescent punctate spots are readily apparent. The proportion of hyperfluorescent cells in L929 (n = 20) and Vero cultures (n = 6) was similar (E), with bars showing standard error. Confocal microscopic analysis of Draq5 (a nuclear stain) and fluorescein-stained L929 cells, revealed the presence of fluorescein throughout the interior of the cell, with numerous highly intense fluorescein-containing structures being visible in the cytoplasm, especially of hyperfluorescent cells (F shows a single confocal ‘slice’ through the cells, and (G) shows the orthogonal view; a 3D reconstruction of staining along the white and yellow axes). Treating control cells with the membrane-slective stain Vybrant ® DiI confirmed the likely appearance after staining with a compound, which localizes on the cell surface, providing further confirmation that fluorescein has entered cells (H). Data shown are representative of several experiments. Scale bars in (A) to (C) represent 100 µm and in (F) to (H) represent 20 µm.</p

Fluorescein and propidium iodine staining of cells treated with MPS or benzalkonium chloride.

<p>L929 cultures were treated with growth medium containing 25% ReNu MultiPlus® MPS or PBS (control) and stained with Hoescht 33342, propidium iodide and fluorescein following an overnight incubation. Typical appearance of control cells is shown after Hoescht 33342 staining (A), propidium iodide staining (B) and fluorescein staining (C), with the equivalents for MPS-treated cells shown in (D) to (F). No correlation was evident between propidium iodide-staining cells and fluorescein hyperfluorescent cells. Notably the number of hyperfluorescent cells was significantly increased after MPS-treatment for both L929 (n = 20 wells) and Vero cultures (n = 6 wells) (p≤0.01) (G). However the overall numbers of PI-staining cells did not increase in L929 cells (n = 20 wells) (H) (this was not tested for Vero cells). Conversely treatment with benzalkonium chloride dramatically increased the number of PI-positive cells, but resulted in no detectable fluorescein hyperfluorescent cells (I). Data shown are representative of several experiments. Scale bars in (A) to (F) represent 100 µm. Bars in (G) to (I) represent standard error.</p