Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Abstract

Source: Liang, K. X., et al. Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives. J. Vis. Exp. (2021)

This video demonstrates the labeling of neuronal cells with antibodies for mitochondrial respiratory chain complexes, mitochondrial DNA-associated protein, and mitochondrial outer membrane protein, followed by flow cytometry to quantify their levels.

Protocol

1. Flow cytometry measurement of MRC (mitochondrial respiratory chain) complex subunits and TFAM (mitochondrial transcription factor A) in fixed cells

1. At the end of the culture period, detach the cells (~10⁶) by adding the cell dissociation reagent; then, pellet and collect the cells in a 15 mL tube. Wash the cells by centrifugation with phosphate buffered saline (PBS) (1x) twice by centrifuging at 300 × g for 5 min.
2. Fix the cells in 1.6% paraformaldehyde (PFA) (1 mL of 1.6% PFA in a 15 mL tube) at room temperature (RT) for 10 min. Wash the cells by centrifugation with PBS (1x) twice by centrifuging at 300 × ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-TFAM conjugated with Alexa Fluor 488	Abcam	ab198308	Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; use mouse monoclonal IgG2b Alexa Fluor® 488 as an isotype control.
anti-TOMM20 conjugated with Alexa Fluor 488	Santa Cruz Biotechnology	Cat# sc-17764 RRID:AB_628381	Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; use mouse monoclonal IgG2a Alexa Fluor® 488 as an isotype control.
anti-NDUFB10	Abcam	ab196019	Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.
anti-SDHA	Abcam	ab137040	Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.
anti-COX IV	Abcam	ab14744, RRID:AB_301443	Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-mouse IgG (1:400, Thermo Fisher Scientific, Catalog # A-11001) as secondary antibody; use mouse monoclonal IgG as an isotype control.
PBS 1x	Thermo Fisher Scientific	18912014	Used for washing steps
Formaldehyde (PFA) 16%	Thermo Fisher Scientific	28908	Cell fixation
Glycine	Sigma-Aldrich	G8898	Used in blocking buffer
Bovine Serum Albumin	Europa Bioproducts	EQBAH62-1000	Blocking agent to prevent non-specific binding of antibodies in immunostaining assays and CDM ingredient