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We suspect that the endogenous (lysosomal) enzyme generates a lot of
fluorescence, since letting cells grow to confluence dramatically increased
that fluorescence. The amount of lysosomal material (and hence enzyme
activity) depend inversely upon cell culture proliferation rates (Poot et
al. (1985) Gerontology 31, 158 -165). J.F. Jongkind (Rotterdam, NL)
pioneered fluorochromatic assays of beta - galactosidase (eg. Cytometry
1986) and found essentially no correlation between enzymatic activity in
homogenates and fluorescence development in an intact cell. Trying to
suppress the lysosomal enzyme activity with chloroquine lead to significant
cell kill. We are curently looking for more specific and non - toxic
inhibitors of this enzyme.
Another problem we encountered was strong leakage of product from the cells,
such that the fluorescence of the staining media exceeded the cellular
fluorescence. This seems to be analogous to the findings of Van Der Velden
et al. (1994), who showed that the conjugate of glutathione with
monochlorobimane gets pumped out of cells. if that also applies to LacZ
assays, we will not be able to improve on those if we do not generate a
product that stays in the cells.
It looks as if we are totally stuck.
Martin Poot
martin@probes.mhs.compuserve.com
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