Measurements were made at 0,6,12,and 22 hours. For each well, a picture was taken at the top-middle and the bottom-middle, for a total of two pictures per well. Three iterations of each concentration( as shown Tables 1-3) gave us 6 data points at each time point for any given concentration of chemical of concern.
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Measurements were made at 0,6,12,and 22 hours. For each well, a picture was taken at the top-middle and the bottom-middle, for a total of two pictures per well. Three iterations of each concentration (as shown Tables 1-3) gave us 6 data points at each time point for any given concentration of chemical of concern.
After the insertion of our promoters into the mammalian cells, we ran a series of initial experiments to optimize our measurement protocol. Initially we measured the fluorescence intensity of our bioassay using a microplate reader. We found that this method resulted in a high autofluorescence from the complete growth media. In order to combat this washed the cells with PBS before testing at each time interval. We found that the PBS wash contributed to cell stress and decreased the viability of the cells. Therefore, we chose to use a fluorescence microscope as our choice for measuring cell fluorescence.
We began testing by assembling our bioassay as shown in the following plate layouts.
Table 1. Plate layout for MT1 promoter construct
Table 2. Plate layout for MT2 full promoter construct
Table 3. Plate layout for MT2 59bp promoter construct
Measurements were made at 0,6,12,and 22 hours. For each well, a picture was taken at the top-middle and the bottom-middle, for a total of two pictures per well. Three iterations of each concentration (as shown Tables 1-3) gave us 6 data points at each time point for any given concentration of chemical of concern.
Once all the pictures were taken, they were analyzed using imageJ. The code was written such that only the parts of the image that display Fluorescence from cells is analyzed. The threshold values used in the code were optimized to reduce noise in the data. The code returns an output of the area of fluorescence analyzed and the mean fluorescence per image. The following imageJ code was used to analyze the images.
title = getTitle();
run("Set Measurements...", "area mean min integrated limit redirect=title decimal=3");
setAutoThreshold("Default”);
//run("Threshold...");
setThreshold(4000, 65535);
run("Measure");
Figure 1 & 2. The picture to the left shows the microscope image captured and the picture to the right shows the result of the image being processed using imageJ.
The data obtained from imageJ was processed using excel. From the data we found that we had to further narrow down the concentration ranges for the chemicals of concern in order to get more defined product to substrate curves. For the second batch of experiments the concentration ranges were further refined and the bioassay was assembled as shown in the following plate layouts.
Table 4. Plate layout for MT1 promoter and MT2 promoter constructs for the second batch of experiments
Table 5. Plate layout for FGF21 promoter and GADD153 promoter constructs for the second batch of experiments
Cenozoic
