Green signal of JC-1 decreased when macrophages underwent damages... do you know why?

29 December 2022 1 6K Report

Hi, I am studying about inflammasome using bone marrow derived macrophages(BMDM).

I used JC-1 dye to measure mitochondrial membrane potential changes caused by damages.

JC-1 can be monomer when mitochondria are damaged and make green fluorescence, and aggregate when mitochondria are health and make red fluorescence.

Commonly, when cells get some stresses, mitochondria are damaged and JC-1 turn to green from red. So, green signal has to increase!

My protocol :

Differentiated BMDM for 6d treated H2O2 and LPS/ATP.

After that, JC-1 treated to final concentration 2 uM for 10 min at 37 degree.

And wash to PBS twice, measured using microplate reader or captured some pictures on microscope.

Sadly, in my results, green fluorescence decreased in damaged samples compared with control samples (non damaged)...

I don't understand how they work...

Are there any persons have problems like this?

Please, help me...

Simon Powell

Probably compromised internal or plasma membranes which caused the monomers to leak out

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