Evinova 2019 MiPschool Coimbra
Event: MiPschool Coimbra 2019
Mitochondrial respiratory functions of the cell models could represent a powerful diagnostic model for evaluation of mitochondrial function in health and disease. Neuroblastoma cell line SH-SY 5Y is widely used in neurodegenerative diseases research as in vitro model. This line is a subline of the SK-N-SH cell line, originated from bone marrow biopsy of a metastatic neuroblastoma of a 4-year old female. Mitochondrial activity in intact SH-SY 5Y and SK-N-SH neuroblastoma cells was determined by using O2k - FluoRespirometer (Oroboros, AT). We tried to characterize and compare mitochondrial activity of both cell lines. Cell lines were undifferentiated. The cells were cultured at 37°, 5% CO2 in DMEM/MEM medium supplemented with 10% fetal bovine serum and 1% of a penicillin/streptomycin stock. This medium was changed at 2–3 day intervals until archieving the 80% confluence. On the analysis day the adherent cells were trypsinized and washed with DPBS twice. Prior 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 3 millions 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. For data analysis, GraphPad Instat was used with Student-Newman-Keuls Multiple Comparisons Test. The data were expressed as mean ±SD. Usage of more than 1 million cells per chamber is sufficient for HRR measurements. We detected that SK-N-SH cells have slightly lower ROUTINE in comparison with their triple cloned SH-SY 5Y cells (Figure 1). We also found that SKN-SH cells have higher spare respiratory capacity compared to SH-SY 5Y cells.
• Bioblast editor: Plangger M
Labels: MiParea: Respiration, Comparative MiP;environmental MiP
Organism: Human Tissue;cell: Neuroblastoma Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET Pathway: N HRR: Oxygraph-2k
Affiliations and support
- Evinová A(1), Hatoková Z(1), Čižmárová B(3), Brodňanová M(2), Račay P(1,2)
- BioMed Martin, Jessenius Fac Medicine Martin
- Dept Medical Biochem, Jessenius Fac Medicine Martin; Comenius Univ Bratislava
- Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik Univ Košice; Slovakia
- This work was supported by the Slovak Research and Development Agency under the contract No. APVV-16-0033.
Figure 1: Representative traces of mitochondrial respiratory function of SH-SY5Y intact cells in MiR05 medium (Oroboros, 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].