Van Dyk 2015 Abstract MiPschool Cape Town 2015

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Optimization of selected cell lines for use on the Seahorse extracellular flux analyzer.

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van Dyk HC, Mereis M, Horak W, Kenosi M, Pretorius M, Louw R, van der Westhuizen FH (2015)

Event: MiPschool Cape Town 2015

The Seahorse XFe96 analyser is able to measure, in real-time and in high throughput, two consequences of in vitro cellular bioenergetics: the oxygen consumption rate (OCR), an indicator of cellular respiration, and the extracellular acidification rate (ECAR), a result of glycolysis. During these processes, several key bioenergetics parameters can be determined in either cell lines or isolated mitochondria. For future investigations on the Seahorse XFe96 analyser, we have selected and obtained from ATCC eight relevant cell lines. These cell lines were: 143B, SH-SY5Y, fibroblasts, H9C2, C2C12, RD, A549 and HEPG2. Each of these cell lines are set to undergo a mitochondrial (or 'mito') stress test in later studies. A mito stress test involves the intermittent injection of oligomycin (ATP synthase inhibitor), FCCP (uncoupler), rotenone and antimycin A (complex I and III inhibitors, respectively) in order to produce a bioenergetic profile. Before any specific experiments could be conducted, we set out to optimize the necessary conditions for each of these cell lines. Four conditions were optimized for each cell line using a basic OCR reaction protocol as provided by the supplier.


Labels: MiParea: Respiration 


Organism: Human  Tissue;cell: Other cell lines, Fibroblast  Preparation: Isolated mitochondria 


Coupling state: LEAK, ROUTINE, ET  Pathway: N, ROX 



Abstract continued

These conditions, along with the concentration ranges used during optimization, were as follows: (1) Cell seeding density per well (on average 5000 - 12500 cells/well in increments of 2500); (2) Oligomycin concentration (0 - 2 M); (3) FCCP concentration (0 - 2 M) (4) Glucose (0 - 25 mM) and pyruvate concentrations (0 - 2 mM) in assay medium. The optimal cell seeding density was determined by the maximal basal respiration OCR values, cell confluency and cell morphology. The optimal cell seeding density per well was found to be between 10 000 and 15 000 cells per well for most cell lines with the exception of fibroblasts, RD and HEPG2 cells which were found to be optimal between 20 000 and 25 000 cells per well. The optimal oligomycin concentration, as determined by maximal ATP production and thus optimal ATP synthase inhibition, was found to be 1 M in all cell lines except the A549 and HEPG2 cell lines where it was found to be 1.5 M. The FCCP concentration varied greatly where it ranged between 0.5 and 1 M in all cell lines except H9C2 where it was found to be 2 M. The optimal FCCP concentration was determined by the highest spare respiratory capacity values that could be attained before OCR values decreased due to excessively decreased membrane potential. In all cell lines the optimal glucose concentration was between 5 and 11 mM with no major difference seen between these two concentrations, while the optimal pyruvate concentration was found to be 1 mM. From these results it could be seen that the cell seeding density varied greatly between different cell lines, presumably due to cell diameter and the growth doubling time of different cell lines. Oligomycin was found to be constant in almost all cell lines while FCCP proved to be much more specific. The optimal pyruvate and glucose concentrations seemed to be 1 mM and 5 mM respectively, but the selection of these concentrations depends on the aim of an experiment and whether a high basal respiration or a high spare respiratory capacity is preferable. As also advised by the supplier, we have concluded from these initial optimization experiments that a thorough optimization of reaction conditions for bioenergetics investigations is essential even if conditions are reported in literature, and will greatly improve the outcomes of subsequent investigations.

Affiliations

Biochem, Centre Human Metabonomics, North-West Univ, South Africa. - hayleyvandyk91@gmail.com