Is exercise-induced mitochondrial degradation necessary for improving mitochondrial health overall?

Hi everyone, I hope everyone can help me with this.

First, I am measuring mitochondrial membrane potential by flow cytometry using TMRE and MTG dyes. I think we are not experiencing problems with my TMRE readings. However, I have problems with my MTG readings. Supposed to be that MTG is independent with the membrane potential. But, if I add uncouplers (CCCP), the values for MTG stained cells are significant lower compared to CCCP untreated samples (30-50% lower).

My procedure is, I stain the cells first with MTG and add the uncoupler (30uM) after 20 mins, and then incubate it for another 10mins (total of 30mins for the dye).

There are 2 possible scenarios that I am thinking.

First, the dye and CCCP interact with each other leading to a lower fluorescence intensity readouts.

or,

Second, the uncoupler cause severe mitochondrial damage that it initiates mitophagy.

but is 10 mins exposure to the uncoupler (plus few more minutes while waiting it to be read in flow cytometer) enough time already to be degraded by mitophagy? or is the first scenario a more logical explanation for the lower values in CCCP treated cells?

Dear RG community,

I have a substance that I want to use it in the cell culture (in vitro). I am using flow cytometry for mitchondrial dynamics and western blotting for mitophagy/autophagy. But I could not decide what dose I should use. The substance is FDA proved and does not harm to human. I started 100uM for some paper suggested (experiment details are all different than mine). I went up to 1mM and the effect that I am getting from flow cytometry is increased gradually but I do not know whether the dose is good or should I go up like 10mM or more? What is the limit and how to determine?

I am planning to do MTT assay 4h 16h 24h 48h 50uM 100uM 500uM 1mM 5mM and 10mM to see the cells growth. But MTT is different experiment and I am investigating different thing. How to combine them to choose proper dose. (By the way what time is more appropriate for MTT?).

I am master student therefore my knowledge is limited please let me know different experiments or protocols to continue. Thanks in advance.

Best

Süleyman

Survival of the species and regulation of heat shock protein (HSP) levels are critical to prevent toxic protein aggregation and mitophagy. Dietary activators, drugs and inhibitors may reverse protein aggregation, but excessive caffeine and curcumin levels may inactivate and promote toxic amyloid beta aggregation. In various species zinc levels and core body temperature are critical to survival with HSP and amyloid regulation important to toxic protein aggregation with relevance to programmed cell death.

Is it possible to applied MitoTimer method on human peripheral blood mononuclear cells?

I want to monitor biogenesis vs mitophagy processes.

If you have any kind of protocol connected I will be happy to get it.

Thanks!!

I stimulated the cells with CCCP (10uM) as well as CCCP + Chloroquine (50uM), as well as having an unstimulated sample. The Mitotracker DR signal massively increased when I added CCCP.

I know the signal is meant to decrease with CCCP, as the mitochondria are removed from the cell via mitophagy, and the addition of chloroquine was meant to prevent their removal.

Any reason why it might have increased?

I used a DAPI live dead solution to only measure the live cells

REFERENCES:

1. Antibiotic Use and Nuclear Receptor Inactivation Linked to Mitophagy in Diabetes and Chronic Diseases. Journal of Diabetes and Clinical Studies. 2018,2 (1);1-3.

2. Sirtuin 1, a Diagnostic Protein Marker and its Relevance to Chronic Disease and Therapeutic Drug Interventions. EC Pharmacology and Toxicology 6.4 (2018): 209-215.

3. Antibiotic Resistance Involves Antimicrobial Inactivation in Global Communities. SAJ Pharma Pharmacol 2017;2: 102.

4. Antimicrobial Drugs and Bacterial Amyloid Peptide Induce Toxic Manifestations in Chronic Diseases. EC Pharmacology and Toxicology 6.1 (2018): 01-04.

5. Bacterial Lipopolysaccharides Change Membrane Fluidity with Relevance to Phospholipid and Amyloid Beta Dynamics in Alzheimer’s Disease. J Microb Biochem Technol. 2016; 8: 322-324.

6. Inactivation of Anti-Aging Genes is Related to Defective Drug Metabolism in Diabetes. Int J Drug Disc. 2017; 1:003.

Hi,

I am starting studying Mitophagy, so I was looking into some publication to see how to measure mitophagy reliably. Like which kind of markers do you usually use?

is colocalization of LC3B and TOMM20 a good strategy?