Characterization
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Problems with cell age In our experiments, we found that whether or not the assay worked is highly dependent on cell age. In the following experiment, we grew cells in liquid medium with PCB in the dark (or DMSO for control), then pipetted an aliquot of cells into each well of a 96 well plate. Each well was exposed to red laser light for the indicated amount of time, and then covered and allowed to incubate for 3 hours. After the incubation period the wells were swabbed with q-tips, which were then dipped in liquid nitrogen to lyse the cells, and then submerged in x-gal solution. Q-tips with cells producing beta gal turned blue.
In this assay, we found that the older cells had higher background in the negative control than the younger cells. This is probably attributable to the cell line we used, Y190. This cell line contains both beta-galactosidase and histidine reporters under the control of the Gal promoter. Because the cells do not typically produce and mutation would give those mutant cells a metabolic advantage. Any mutations in that would upregulate His production could also lead to an increase in constitutive beta-gal expression. We attempted to reduce this effect by supplementing our media and plates with additional His, but this hypothesis remains untested and further exploration is needed.
Problems with concentration We also experienced problems with cell concentration: If cell concentration was too high, we saw significant positive background. This is probably due to low levels of production of beta gal in the absence of induction, which is magnified as cell concentration increases, resulting in high positive background on controls if the cells used are too concentrated. Although it is preferable to not allow the cells to overgrow, we have evidence that dilution of a culture works to help reduce positive background.
In this assay, we exposed cells to light or dark according to the following diagram, doing sequential dilutions to examine the effects of cell concentration. Although at the highest concentration there was significant background on the negative control, at a 10 fold dilution, the background is reduced significantly. Further dilutions are less effective because the blue becomes too difficult to see.
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