Team:Hong Kong HKUST/Safety

Overview

Since the project measures fatty acid (FA) up take rate at different fatty acid concentrations, cell viability was measured to assure that cells are stable and alive in various FA concentrations. The change of FA uptake rate is expected to be affected by both the inducible and constitutive glyoxylate shunt transfected into human liver cells, respectively.

Cell Line

HepG2 cells were used for testing constitutive and inducible glyoxylate shunt by measuring change in sodium palmitate uptake rate by the cell. Ultimately, we are testing the glyoxylate shunt inside HepG2 cells, thus it was subject of study in our cell viability test.

Cell Viability

To make sure the whole system we created works well in cells, we also have to use MTT assay to test cell viabilities in different FA concentrations. The optimal goal is to determine the range of optimal concentrations of fatty acids to introduce into HepG2 cell and achieve more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.

MTT assay description

Cell viability was measured to identify the amount of sodium palmitate that we need in the medium to do our characterization, including to make sure that the enzymes are expressed stably inside cells. High concentration of sodium palmitate can induce cell death. We, therefore, had to determine the concentration of sodium palmitate that is high enough to activate our promoter, but not excessive to kill our cells.
MTT assay was used to measure the cell viability. It measures the enzymatic activity of oxidoreductase enzymes that only show activity when the cells are alive. MTT, a tetrazolum dye, is redued into an insoluble formazan , giving a purple color. Organic solvent such as DMSO can be used to dissolve the formazan. Absorbance at 570 is measured using a spectrophotometer to quantitatively determine the amount of formazan formation.
In our experiment, HepG2 cells were seeded into a 96-well plate. After one day incubation gradient concentration of sodium palmitate from 0 mM to 1.0mM, and 2.0mM were added into each row. After adding the sodium palmitate, we have incubated the cells for 24 hours and 48 hours. MTT reagent was added and formazan formation was observed and measured.

Results

From the MTT assay, we can conclude that after 24 hours of incubation with palmitic acid, around 45% of cell viability can be maintained even at 2.0mM. For 48 hours incubation, cell viability is very different. To maintain 50% cell viability after 48 hours incubation with palmitic acid, we cannot exceed 0.32 mM of palmitic acid.

Transfection and Fatty Acid Uptake Rate

The ACE proteins for glyoxylate shunt were fused with fatty acid-induced promoters. The promoters were induced by introducing variable concentrations of sodium palmitate to trigger greater expression. To observe the difference in fatty acid uptake rate for constitutive and inducible system where enzymes expression were triggered, we also did a parallel transfection of BioBrick containing constitutive promoter to see the variations of FA uptake rate.
To get the uptake rate of FA, we decided to quantify FA concentration in medium time by time. We are right now making our determination to use FA quantification assay kit, Sigma Fatty Acid Quantification Kit from TIN HANG TECHNOLOGY LIMITED.

Transfection Method