Flow cytometric analysis of mitochondrial electrochemical potential

Flow cytometric analysis 

• Neurons DRGs were cultured for 48h with Chimera C or Vehicle. 
• DRGs were stained in situ with TMRE (200 nM, Invitrogen Thermo Fisher Scientific Cat:# T-669) for 30 min at 37°C in 5% CO₂–95% air, 
• washed twice in cool-PBS 
•  0.05% Trypsin-EDTA was added to release DRGs (Gibco, cat:# 1995647). 
•  Centrifugation 2.500 RPM for 5 mins 4°C
•  DRGs pellets were re-suspended in 200 µl of FACS buffer (PBS 1X, BSA 1X, 2 Mm EDTA). 
• Flow cytometry of TMRE fluorescence was performed on a Gallios instrument (Beckman Coulter) and analyzed using FlowJo10 software. ~3500 events were acquired for each sample. 
• Data are presented as Mean Fluorescence Intensity per experiment. 
• Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 10 μM for 1 hr) (Sigma, Cat #C2759) was added as a positive control for mitochondrial depolarization.

Reagents:

• TMRE (200 nM, Invitrogen Thermo Fisher Scientific Cat:# T-669)
• 0.05% Trypsin-EDTA (Gibco, cat:# 1995647)
• Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 10 μM for 1 hr) (Sigma, Cat #C2759)

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