Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Take fixed and permeabilized neurons in a tube.

Add a blocking buffer to prevent non-specific antibody binding.

Centrifuge the suspension and remove the supernatant. Then, resuspend the neurons in buffer.

Transfer the suspension equally into tubes containing untagged and fluorophore-tagged primary antibodies.

Incubate, allowing the antibodies to specifically bind to mitochondrial respiratory chain or MRC complex subunits, a mitochondrial DNA-associated protein, and a mitochondrial outer membrane protein within the neurons.

Remove any unbound antibodies.

Incubate with fluorophore-tagged secondary antibodies that bind to the untagged primary antibodies.

Centrifuge the mixture and discard the supernatant.

Resuspend the neurons in buffer and transfer them to microcentrifuge tubes.

Perform flow cytometry to detect the fluorescent signals on the labeled neurons, corresponding to the expression levels of MRC complex subunits and the measurement of relative mitochondrial DNA levels.