Categorías y Variables Económicas

Initial dose-finding experiments investigating the effect of EPA on HUVEC proliferation were performed using a 96-well plate with 6 replicates for each cell condition (Experiments 1 and 2). There were no viable cells seen at 24hrs when cells were supplemented with EPA concentrations greater than 50µM, and MTT optical density readings at these concentration were equivalent to the no-cell controls (Figures 6.1 and 6.2). This suggested that EPA ≥ 50µM was cytotoxic to cells. There appeared to be a dose-dependent effect of EPA on HUVEC proliferation between EPA 5-50µM. In Experiment 2, duplicate 96-well plates were seeded. In one, culture media was left unchanged for 72hrs, and in the other, culture media in each well was changed after 48hrs. Changing the culture media at 48hrs was associated with increased proliferation across all cell conditions compared with not changing the culture medium (Figure 6.2). However, because this affected all cell lines equally it was decided for future experiments not to change the culture media during the experiment in order to a) simplify the experiment, b) minimise wastage of expensive culture medium, and c) minimise error in repeatedly making up small volumes of EPA containing media.

The assay was then scaled up to a 48 well format to increase the number of cells per well and allow better visualisation of the cells under microscopy (Experiment 3).

The dose range of EPA was narrowed to 0-30µM. Similar to the 96-well format, a dose-dependent reduction in HUVEC cell proliferation was observed (Figure 6.3).

Figure 6.1. The effect of EPA on HUVEC proliferation: Experiment 1. Each data point is the mean of 6 replicate wells.

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24 hr 48 hr 72 hr 96 hr

Optical Density

Effect of acute EPA supplementation on HUVEC proliferation Experiment 1

no cell control media control media+vehicle control EPA 5

EPA 10 EPA 25 EPA 50 EPA 100

Figure 6.2. The effect of EPA on HUVEC proliferation: Experiment 2.

In experiment 2a culture media was left for the duration of the experiment, whereas in 2b the culture media in each well was aspirated and replaced with fresh medium at 48hrs.

Each data point is the mean of 6 replicate wells.

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Effect of acute EPA supplementation on HUVEC proliferation Experiment 2a

Effect of acute EPA supplementation on HUVEC proliferation Experiment 2b

Figure 6.3. The effect of EPA on HUVEC proliferation: Experiment 3. In this experiment the effect of EPA 5-30µM was investigated in a 48-well format. Each data point is the mean of 12 replicate wells.

After scaling up the experiment to the 48-well format and observing similar results to the 96-well format, it was decided to validate the MTT assay before performing further experiments in the 48-well format. The purpose of this was two-fold. Firstly, to confirm that the MTT assay OD readings were proportional to cell number, and secondly to exclude the possibility that EPA had a direct effect on the assay itself by interfering with mitochondrial metabolism of MTT and the solubilisation of formazan crystals by propan-1-ol.

To investigate whether MTT assay OD readings were proportional to cell number.

serial 2-fold dilution of cells were plated at densities of 0 - 32000 cells/well and incubated for 24hrs before performing an MTT assay. Figure 6.4 demonstrates that OD was proportional to seeded cell number. This relationship was linear up to a

Effect of acute EPA supplementation on HUVEC proliferation Experiment 3

Figure 6.4. Optical density vs. seeded cell number. Serially diluted HUVECs were plated and allowed to attach for 24hrs. MTT assay was then performed and optical density plotted against seeded cell number. A line of best fit has been added between cell densities of 0-16000 cells. Each data point is the mean of 6 replicate wells.

To investigate whether EPA directly affects the MTT assay rather than affecting cell proliferation, 1x10⁴ cells were incubated in plain culture medium for 24hrs. EPA 100µM or an equivalent volume of ethanol carrier were added to one row each at the same time as adding the MTT to the cultured cells, and again to separate rows at the time of solubilising the formazan crystals with propan-1-ol. These experimental conditions are summarized in Table 6.1.

Figure 6.5 demonstrates that the addition of ethanol carrier had no effect at any stage of the MTT assay. Addition of EPA at the MTT step caused a reduction in OD, whereas addition of EPA at the propan-1-ol step had no effect on OD. Under microscopy (Figure 6.6) it was clear that the cells which received MTT+EPA were less viable than those which received MTT alone, and resembled the cells seen previously when grown in media supplemented with EPA50-100µM. It is therefore likely that 100µM EPA had a cytotoxic effect on cells within the 3 hours incubation

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0 4000 8000 12000 16000 20000 24000 28000 32000

Optical density

Cell number

Optical density vs. seeded cell number

period when added with the MTT, rather than EPA directly affecting the performance of the assay by preventing the metabolism of MTT by viable cells.

Culture

Table 6.1. Experimental conditions for each row of the 48-well plate. These conditions were replicated in 6 wells per row.

Figure 6.5. Effect of EPA and ethanol (EtOH) carrier on the performance of the MTT assay. Each data point represents a single value.

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Effect of EPA and ethanol carrier on the MTT assay

No-cell control

Figure 6.6. Representative photos of cells seeded in a 48-well plate. Photograph of cells (x10 magnification) remaining in wells after MTT incubation, from wells treated with (a) MTT alone as per the standard assay and (b) MTT + 100µM EPA.