Evinova 2019 MiPschool Coimbra

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High resolution respirometry of neuroblastoma SK-N-SH and SH-SY 5Y cell lines.

Link: MitoEAGLE

Evinova A, Hatokova Z, Cizmarova B, Brodnanova M, Racay P (2019)

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 37ยฐ, 5% CO2 in DMEM/MEM medium supplemented with 10% fetal bovine serum and 1% of a penicillin/streptomycin stock. This medium was changed at 2โ€“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 ยฑSD. 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.

โ€ข 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:HRR: Oxygraph-2k 

Affiliations and support

Evinovรก A(1), Hatokovรก Z(1), ฤŒiลพmรกrovรก B(3), Brodลˆanovรก M(2), Raฤay P(1,2)
  1. BioMed Martin, Jessenius Fac Medicine Martin
  2. Dept Medical Biochem, Jessenius Fac Medicine Martin; Comenius Univ Bratislava
  3. Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef ล afรกrik Univ Koลกice; Slovakia
This work was supported by the Slovak Research and Development Agency under the contract No. APVV-16-0033.


Evinova Figure1.jpg

Figure 1: Representative traces of mitochondrial respiratory function of SH-SY5Y intact cells in MiR05 medium (Oroboros, Innsbruck AT) at 37 ยฐC with continuous stirring (speed 750 rpm). The blue line represents oxygen concentration [ฮผM] and the red line represents oxygen consumption as oxygen flow per cells IO2 [pmol O2 s-1 10-6 cells].

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