Markova 2016 Abstract MitoFit Science Camp 2016

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Porcine hearts samples preparation: PBI-Shredder or saponin?

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Markova M, Cedíkova M, Grundmanova M, Kuncova J (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

High-resolution respirometry is currently one of the key methods for mitochondrial respiratory function analysis. Success of this method strongly depends on the sample preparation in the very beginning of the experiment. Our aim was to optimize the heart sample preparation from experimental pigs with minimal mitochondrial damage in the sample and sufficient penetration of the substrates utilized during respirometry.

Hearts from experimental pigs were excised and placed in ice-cold Ca2+ free Tyrode solution. Free wall of the left ventricle was excised from the heart and was inserted into a Falcon tube with ice-cold BIOPS. Approximately 2mg of sample tissue were cut off on ice from the left ventricle and were processed by two different protocols. One half of the samples was homogenized using the PBI-Shredder [1] (metal lysis disk; 10/5s), whereas the second half of the samples was permeabilized with saponin (30 minutes permeabilization in BIOPS with saponin [2] without prior mechanical separation, followed by 10 minutes rinsing in MIR05). Prepared samples were measured in Oroboros Oxygraph-2k chambers with appropriate SUIT protocol (GMP+D+c+Rot+S+Ama+As+Tm+Azd) in mitochondrial respiratory medium MIR06 [3]. Oxygen concentration was increased to around 400 µM by titrating an appropriate amount of H2O2. Acquired mitochondrial respiratory parameters were analyzed and compared.

Major difference in respiration between two types of sample preparation methods (PBI-Shredder or saponin) was observed upon addition of cytochrome c. In homogenates prepared with PBI-Shredder mean increase of respiration upon addition of cytochrome c was 26%. Mean increase of respiration after cytochrome c addition in samples permeabilized with saponin was 14%. On the other hand this respiration increase in samples prepared with the PBI-Shredder showed less variability than increase in samples prepared with saponin. In conclusion, saponin permeabilization seems to be better method for porcine heart samples preparation. Smaller respiration increase after addition of cytochrome c indicates less loss of mitochondrial outer membrane integrity in these samples.

Keywords: PBI-Shredder

O2k-Network Lab: CZ Pilsen Kuncova J


Labels: MiParea: Respiration, Instruments;methods 


Organism: Pig  Tissue;cell: Heart  Preparation: Permeabilized tissue, Homogenate 


Pathway: N, S, NS  HRR: Oxygraph-2k  Event: D1  MitoFit Science Camp 2016 

Affiliations

1-Inst Physiol; 2-Biomedical center; LF UK Pilsen, Czech Republic. - Michaela.Markova@lfp.cuni.cz

References

  1. Draxl A, Eigentler A, Gnaiger E (2013) PBI-Shredder HRR-Set: preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 17.02:1-9.
  2. Canto C, Garcia-Roves PM (2015) High-resolution respirometry for mitochondrial characterization of ex vivo mouse tissues. Curr Protoc Mouse Biol 5:135-53.
  3. Fasching M, Fontana-Ayoub M, Gnaiger E (2014) Mitochondrial respiration medium - MiR06. MitochondrPhysiol Network 14.13:1-4.