I'm trying to find MMP of isolated chicken hepatocytes Mitochondria Infected with Salmonella Typhimurium using JC-1 dye.
I want to ask, Do i have to use fresh isolated Mitochondria from Liver tissues every time or i can store it at -80 degrees.?
or i should shift myself to use single cell suspension and Flow cytometry technique instead of using isolated Mitochondria?
Secondly is there any parameter to check the viability of isolated Mitochondria or after isolation of Mitochondria i Can directly do JC-1 Staining? and How to Calculate red/green fluorescence ratio of isolated Mitochondria after JC-1 treatment
I'll be very grateful
Thank you.
Dear SAajjad,
You should not store mitochondria; they are quite sensitive and isolate intact and pure mitochondria is already hard...
I isolated mitochondria (from HeLa cells) and cecked the integrity and evaluated the mitochondrial membrane potential as marker of functionality.
Also you should run WB to confirm the purity.
You may check my paper "Analysis of hydrophobic and hydrophilic moments of short penetrating peptides for enhancing mitochondrial localization: prediction and validation" for you reference.
Best,
Marco.
Hi
(1)When you thaw your mitochondrial particles, most of them will be dead and it will be practicality impossible to see the ratio of dead/ative ones, especially vy JC-like reagents. To keep organelles alive for about a day, you better store them at 2-4C in a substrate-rich buffer. (2) Measuring respiration is the only reliable method to make sure mitochondria are active. Even ATP synthesis is not a good option as you will see some residual activity. So a small cheap polarograph should help.
Thank you Alex for your kind response, your suggestions are very helpful for me.
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