Coxito 2019 MiPschool Coimbra

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An approach to mitochondrial swelling in optical density microplate assays.

Link: MitoEAGLE

Coxito PM, Stevanovic J, Beleza J, Ascensao A, Magalhaes J (2019)

Event: MiPschool Coimbra 2019

COST Action MitoEAGLE

Mitochondrial permeability transition (MPT) and its relationships with apoptosis and cellular calcium homeostasis, has been an area of devoted research in the last few decades. Numerous and different protocols have been developed or adapted to this subject. One simple, old and common assay relies on the negative correlation between mitochondrial volume and light scattering of a mitochondrial suspension. Measurement of optical density of a mitochondrial preparation at 540nm can follow mitochondrial swelling after MPT pore opening.

However, the usual protocols using cuvette photometers can be both time consuming and sample “expensive”. Microplate reader swelling protocols have been developed [1, 2] with some obvious advantages, specially studying drug dose-response. Yet the practice is not well standardized and there are some pitfalls that arise from having no stirring, long wait times between time points and more data to process.

Here we propose a microplate protocol that even though is based on others [1-3] minimizes these shortcomings and tries to improve analysis rigor.

Based on this experimental approach, we have established that a good resuspension is essential to avoid artifacts due poor homogenization of compounds added to mitochondria in each plate well. Moreover, fast additions and small time point intervals seem to be critical in some assay conditions to be able to record valuable initial data. Automatizing data import, establishing parameter acquisition with low manual input, are important tools to reduce error and bias, by pre-establishing analysis criteria easily kept between all samples. Ultimately, the use of good data filters (Savitzky-Golay) is an important part in the construction of these tools not only because of easier graphical inspection of some calculated parameters but also because it makes it easier to calculate/identify other parameters through derivatives.


Bioblast editor: Plangger M


Labels: MiParea: Instruments;methods 

Stress:Permeability transition 






Affiliations and support

LaMetEx — Lab Metabolism Exercise, CIAFEL — Research Centre Physical Activity, Health Leisure, Fac Sport, Univ Porto, Portugal
Funding: SFRH/BD/129645/2017;SFRH/BPD/116061/2016;UID/DTP/00617/2013;POCI-01- 0145-FEDER-016690-PTDC/DTP-DES/7087/2014;POCI-01-0145-FEDER-016657- PTDC/DTP-DES/1082/2014;EU’s Horizon 2020 Research and Innovation programme Marie Skłodowska-Curie (No.722619,FOIE GRAS;No.734719,mtFOIE GRAS).

References

  1. Waldmeier PC, Feldtrauer JJ, Qian T, Lemasters JJ (2002) Inhibition of the mitochondrial permeability transition by the nonimmunosuppressive cyclosporin derivative NIM811. Mol Pharmacol 62:22-9.
  2. Marroquin L, Swiss R, and Will Y (2014) Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria. Curr Protoc Toxicol 60:25.4.1-17.
  3. Moon SH, Liu X, Cedars AM, Yang K, Kiebish MA, Joseph SM, Kelley J, Jenkins CM, Gross RW (2018) Heart failure-induced activation of phospholipase iPLA2gamma generates hydroxyeicosatetraenoic acids opening the mitochondrial permeability transition pore. J Biol Chem 293:115-29.