Therefore the apparent high levels of fluorescein in hyperfluorescent cells would need to be confirmed by other methods to be certain. Significantly, we found that treatment with the MPS used in this work causes an increase in the proportion of hyperfluorescent cells. These data support the hypothesis that cellular accumulation of fluorescein may underlie clinical observations of fluorescein staining, at least for SICS. This does not exclude the possibility that other interactions of fluorescein with corneal tissues may also contribute, for example fluorescein pooling or preservative-associated transient hyperfluorescence. Additionally we did not examine the effect of cellular confluency on this, which would be an interesting aspect to explore in future studies. Notably we did not find evidence that MPS-treatment of cells caused intense fluorescein staining on the cell surface, as may have been expected, should preservative interactions with fluorescein be responsible for fluorescein hyperfluorescence as shown by Bright et al. Gentiopicrin However it is unclear whether the levels of the preservative in our experimental models would reach those Tubuloside-A likely to be encountered at the cornea, and so further work is needed to establish the role of this potential mechanism in vivo. Importantly, we did not find any association between cell hyperfluorescence and cell death for control and MPS-treated cell populations. We therefore conclude that hyperfluorescence is not a simple cell biological marker for the end-stage of cell death, at least for our two diverse cell types. Furthermore, we found that fluorescein uptake in living cells is dependent on those cells being intact, as cells deliberately lysed by benzalkonium chloride exhibit minimal levels of hyperfluorescence. These data strongly suggest that the hyperfluorescence observable during SICS does not simply reflect increased numbers of dead cells being present, in response to the presence of MPS. Consistent with this is our observation that both fluorescein uptake and release are profoundly influenced by temperature, indicating that these processes involve active transport in living cells. It should be noted that one previous study has reported that the polarization of fluorescein is affected by temperature ; however the ArrayScan II system we used in temperature experiments does not make use of polarizing filters, and so it is likely that the changes we measured in numbers of hyperfluorescent cells incubated at different temperatures genuinely reflects cellular changes in fluorescein levels. These data further support our findings that the fluorescein is distributed throughout the cell, is concentrated by living cells, and does not accumulate preferentially in lysed or dead cells. These findings may also be relevant to the use of fluorescein in cell biological research as a marker for other substances, as our data indicate that any inadvertently non-conjugated fluorescein contaminants are likely to be taken up by living cells, potentially introducing artifacts. Together these data suggest that the increased number of hyperfluorescent cells seen after exposure to MPS reflect an imbalance between active uptake and release processes for fluorescein occurring in these cells. We cannot however exclude the possibility that adverse events such as apoptosis occur in the hyperfluorescent cells, which would not impact the membrane integrity, but which might change the balance between the rate of fluorescein entry and exit. The finding of poor association of hyperfluorescence with end stage cell death is of interest in interpreting the broader clinical significance of SICS and other forms of fluorescein.