Mitotracker dyes are added to the culture medium and incubated for some time to stain mitochondria. I wonder if these can also stain isolated mitochondria? Also is there any specific buffer requirement or cell fractionation buffer will suffice?
Dear Sameer!
Please You look at the article:
Article Measurement of Mitochondrial Mass by Flow Cytometry during O...
2.2. Mitochondrial Isolation
Treated PBL were washed twice with phosphate-buffered saline (PBS) before beginning the mitochondrial isolation. Mitochondria were isolated using a kit purchased from Pierce (Thermo Scientific Pierce, Rockford, IL, USA; catalog No. 89874)
2.3. Flow Cytometry Analysis
All flow cytometry experiments were performed on a BD LSR II (BD Biosciences, San Jose, CA, USA). The fluorescent probes were purchased from Invitrogen/Molecular Probes (Eugene, OR, USA) unless otherwise stated. ΔΨm was measured using 10 nM tetramethylrhodamine, methyl ester (TMRM) (catalog No. T668; ex543, em567) and 40 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6) (catalog No. D273; ex488, em525). Mitochondrial mass was evaluated with 150 nM MitoTracker Green (MTG) (catalog No. M7514; ex490, em516) and 2.5 μM nonylacridine orange (NAO) (catalog No. A1372; ex490, em540). The concentration of NO, a reactive nitrogen species (RNS), was measured by 1 μM 4-amino-5-methylamino-2′,7′-difluorescein (DAF-FM) (catalog No. D23844; ex495, em518). H2O2levels were evaluated using 10 μM 2′,7′-dichlorofluorescin diacetate (DCF-DA) (catalog No. C400; ex495, em529). Dihydrorhodamine 123 (DHR) (catalog No. D23806; ex507 em527) and dihydroethidium (HE) (catalog No. D11347; ex635 em610) were also used. Data were analyzed with FlowJo version 7.5.5 software (Tree Star Inc., Ashland, OR, USA).
Dear Chernov,
Thanks for sharing the article. It is very informative.
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