This article describes a protocol for analyzing mitochondrial respiratory chain complex subunits in fixed and permeabilized neurons using flow cytometry. The method involves antibody labeling to detect specific mitochondrial proteins and assess mitochondrial DNA levels.
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.
Block the samples in 1 milliliter of blocking buffer, and wash the cells by centrifugation with PBS as demonstrated. To detect different mitochondrial complexes and subunits, incubate the cells for 30 minutes with the respective primary antibodies described in the text manuscript. After washing the cells once with PBS, incubate the cells with secondary antibody for 30 minutes.
At the end of the incubation, wash and resuspend the cells in PBS as demonstrated. Transfer the cell suspension into a 1.5-milliliter microcentrifuge tube, kept in the dark, on ice. Next, analyze the cells on the flow cytometer and detect signals and filter one using a 530/30 bandpass filter.
繁體中文
