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Team:Hong Kong HKUST/experiment/exp1
From 2013.igem.org
Cell Viability & Fatty Acid Quantification
Cell Viability
Cell Culture and Transfection
We cultured HEK293FT cells following the American Type Culture Collection’s standard procedure, except that we have used DMEM with 10% FBS and 1% penicillin/streptomycin for our culture medium. For transfection, we followed the manufacturer’s protocol of LipofectamineTM 2000 (Invitrogen). We used serum-free and antibiotics-free DMEM to form a DNA-lipofectamine complex.
Cell Line
In our project, two mammalian cell lines were used: human hepatoma cell (HepG2 cell) and human embryonic kidney 293 cell (HEK293FT). HepG2 cells were used for characterizing inducible promoters and glyoxylate systems. For higher transfection efficiency, the characterizations of mitochondrial leader sequence and constitutive promoter CMV were conducted using HEK293FT cells.
Cell Culture
HepG2 and HEK293FT cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 50ug/mL penicillin, 50ug/mL streptomycin at 37℃in a humidified atmosphere containing 5% CO2. Cells were transfected in petri dishes and multi-well plates with different construct Lipofectamine 2000 (Invitrogen; Carlsbard, CA) according to manufacturer’s protocols. Any GFP signals were observed under fluorescent microscope or under confocal microscope if necessary.
Cell Viability
We designed to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. Fatty acids uptake was to be quantified to compare the activities of wild type cells and cells expressing inducible glyoxylate shunt.
To facilitate expression of inducible glyoxylate shunt in human hepatoma cell line (HepG2 cell), cell viability at different sodium palmitate concentration was measured. While a high fatty acid level is known to lead apoptosis, the cell viability test ensured maintenance of a stable cell line for transfection.
We used MTT assay to test cell viabilities in different fatty acid concentrations. The objective was to determine a range of optimal concentrations of fatty acids to be introduced into HepG2 cell and achieve more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.
MTT Assay Description
MTT assay measures the enzymatic activity of oxidoreductase enzymes that only show activity when the cells are alive. MTT, a tetrazolum dye, is reduced 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 respectively. MTT reagent was added and formazan formation was observed and measured using spectrophotometer.
Results
Figure 1: Average Cell Viability in Different Palmitic Acid Concentrations. A. HepG2 cells were incubated in gradient concentration of sodium palmitate from 0mM to 1.0mM and 2.0mM for 24hours. MTT reagents were added and formazan formation was measured using spectrophotometer. The relative ratio of absorbance at different fatty acid concentration was then calculated by comparing with A570 at 0mM fatty acid concentration. The percentage of relative absorbance ratio was regarded as values of cell viability. B. HepG2 cells were incubated in gradient concentration of sodium palmitate for 48hours.
From the MTT assay, we observed that after 24 hours of incubation with palmitic acid, around45% of cell viability can be maintained even at 2.0mM. For 48 hours incubation, cell viability varied for different concentrations. To maintain 50% cell viability after 48 hours incubation with palmitic acid, we concluded to experiment within range from 0mM to 0.32 mM of palmitic acid.
Fatty Acid Quantification
Quantification Method
Two fatty acid quantification methods were investigated to measure fatty acid uptake rate ofconstitutive and inducible glyoxylate system: 1) Gas Chromatography-Mass Spectrophotometry (GC-MS), and 2) Fatty acid quantification kit (Sigma Aldrich). While we managed to measure fatty acid amount in cell culture medium using GC-MS, fatty acid quantification kit could not be tested due to limitation of time.
Fatty Acid Treatment
In measuring fatty acid, fatty acid solution was mixed with ethanol and chloroform. After acidifying by HCl and refluxing in water bath for 30 min, the organic layer containing fatty acid was collected and extracted by diethyl ether and petroleum ether solution. Again the organic layer was sucked out to be dried before NaOH was added. Then after derivatisation by BF3 and bromotetradecane, the organic layer was collected into GC-MS vial for analysis.
GC-MS
GC-MS is a very useful tool to quantify volatile compounds effectively. For our experiment, we conducted calibration test using known concentrations of fatty acids. However, it was difficult to reach a conclusion due to lack of internal standards and uncertain amount of sample loss.
