Evinova 2018 MiP2018
For characterization of mitochondrial activity in intact SH-SY5Y neuroblastoma cells high-resolution respirometry (HRR) measurements were performed in two chamber system O2k-FluoRespirometer (Oroboros Instruments, AT), by using mitochondrial respiration medium MiR05-Kit (Oroboros Instruments, AT) at 37°C with continuous stirring (speed 750 rpm). We tried to optimize the amount of cultured cells per chamber for further experiments. We have used undifferentiated SH-SY5Y cells. The cells were cultured at 37°, 5% CO2 in DMEM medium supplemented with 10% fetal bovine serum and 1% of a penicillin/streptomycin stock. This medium was changed at 2–3 day intervals until achieving the 80% confluence. On the analysis day the adherent cells were trypsinized and washed with DPBS twice. Prior to the measurement the cells were counted using automated cell counter (Invitrogen) and cell staining with trypan blue. Experiments were performed on intact cell in concentration from 1 to 4 million cells per chamber, repeated three times. Coupling control protocol (CCP) was used for a measurement of intact cells respiration in different coupling control states including ROUTINE, LEAK and ET-pathway. We also checked the coupling capacity of SH-SY5Y cells in the absence or presence of low (0.25 μM) and high (2.5 μM) oligomycin. For data analysis, GraphPad Instat was used with Student-Newman-Keuls Multiple Comparisons Test. The data were expressed as mean ±SD. We detected that 2 milions cells per chamber is enough for HRR measurements of SH-SY5Y cells (Figure 1). We did not observe the statistic significant difference in the coupling capacity using higher or lower concentration of oligomycin.
Labels: MiParea: Respiration Pathology: Cancer
Organism: Human Tissue;cell: Other cell lines, Neuroblastoma Preparation: Intact cells
Regulation: Threshold;excess capacity Coupling state: LEAK, ROUTINE, ET
Evinová A(1), Cizmarova B(3), Pilchová I(1), Brodňanová M(2), Račay P(1,2)
- BioMed Martin
- Dept Medical Biochem; Jessenius Fac Medicine Martin, Comenius Univ Bratislava
- Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik Univ Košice; Slovakia. - [email protected]
Figure 1: Representative traces of mitochondrial respiratory function of SH-SY5Y intact cells in MiR05 medium (Oroboros Instruments, Innsbruck AT) at 37 °C with continuous stirring (speed 750 rpm). The blue line represents oxygen concentration [µM] and the red line represents oxygen consumption as oxygen flow per cells IO2 [pmol O2 s-1 10-6 cells].
This work was supported by the Slovak Research and Development Agency under the contract No. APVV-16-0033.