Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Protocolo

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 × g for 5 min.
3. Permeabilize the cells with ice-cold 90% methanol (1 mL of 90% methanol in a 15 mL tube) at -20 °C for 20 min.
4. Block the samples in blocking buffer containing 0.3 M glycine, 5% goat serum, and 1% bovine serum albumin (BSA) - Fraction V in PBS (1x) (1 mL of blocking buffer in a 15 mL tube). Wash the cells by centrifugation with PBS (1x) twice.
5. Incubate the cells with the following primary antibodies for 30 min: anti-NDUFB10 (1:1,000) for measurement of complex I subunit, anti-succinate dehydrogenase complex flavoprotein subunit A (SDHA, 1:1,000) for measurement of complex II subunit and anti-COX IV (1:1,000) for measurement of complex IV subunit, and anti-TFAM (mitochondrial transcription factor A) antibody conjugated with Alexa Fluor 488 (1:400). Stain the same number of cells separately with anti-TOMM20 (translocase of outer mitochondrial membrane 20) antibody conjugated with Alexa Fluor 488 (1:400) for 30 min (1 mL of primary antibody solution in a 15 mL tube; see the Table of Materials for details about the antibodies).
6. Wash the cells with PBS (1x) once with centrifugation at 300 × g for 5 min. Add secondary antibody (1:400) into tubes of NDUFB10, SDHA, and COX IV and incubate the cells with these solutions for 30 min.
7. Wash the cells with PBS (1x) once by centrifuging at 300 × g for 5 min. Aspirate the supernatants, leaving approximately 100 µL in the tubes. Resuspend the cell pellets in 300 µL of PBS (1x). Transfer the cells to 1.5 mL microcentrifuge tubes kept in the dark on ice.
8. Analyze the cells on the flow cytometer (with a 3 blue and 1 red laser configuration). Detect signals in filter 1 (FL1) using a 530/30 bandpass filter.

Materiales

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