Assessing Mitochondrial Function in Cerebral Vascular Endothelial Cells

Abstract

Source: Rellick, S. L. et al. Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells. J. Vis. Exp. (2016)

This video demonstrates the assessment of the mitochondrial functioning of cerebral vascular endothelial (CVE) cells using an extracellular flux assay. CVE cells are plated in an extracellular flux cell culture plate. Extracellular flux sensors are hydrated before loading and calibration into the bioanalyzer. The plate containing cells is then loaded for analysis.

Protocol

1. Seed and Treat Cerebral Vascular Endothelial (CVE) cells on Cell Culture Plates

1. Seed CVE cells (bEnd.3 cells) on a 96-well extracellular flux cell culture plate: 80 µL/well. Place the cell culture plate at 37 °C in a 5% CO₂ incubator. Note: 16 × 10³ cells per well will reach 60 - 80% confluency within 24 h.
2. If the cells are being exposed to a chemical, add the treatments once the cells are attached to the plate bottom (8 - 24 hours after seeding).
   NOTE: For transfection experiments, do not add antibiotics in a complete growth medium.

2. Assessment of Mitochondrial Function Using the Bioanalyzer

1. Prior to the start of the assay, hydrate an extracellular flux sensor cartridge with calibrant by adding 150 µL of extracellular flux calibrant solution to the plate and incubating overnight at 37 °C without CO₂.
2. Using the plate washer station, change the cell culture medium into pH 7.0 extracellular flux assay medium. Incubate the cells at 37 °C without CO₂ for 30 - 60 min.
3. Prepare working solutions of 8 µM oligomycin, 4.5 µM Carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 10 µM rotenone, and 10 µM antimycin A in Dulbecco's Modified Eagle's Medium (DMEM). Load 25 µL of the stock solutions into cartridge injection ports: Port A, oligomycin; Port B, FCCP; Port C, rotenone, and antimycin A with extracellular flux assay medium. Implement the assay protocol as described in Table 1.
4. Load the hydrated cartridge into the bioanalyzer and perform the calibration by clicking the "Start" button. After the calibration is complete, remove the cartridge bottom plate and load the cell plate by clicking the "Unload Cartridge" prompt. Continue the assay until all measurements are completed.
5. Open the data file and obtain the rate values from the bioanalyzer. Export the data to a spreadsheet file.

Table 1. The Instrument Run Protocol.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
bEnd.3 cell line	ATCC	CRL-2299	25-30 passages
Dulbecco's Modified Eagle's Medium (DMEM)	ATCC	30-2002
Fetal bovine serum	Atlanta Biologicals	S12450	10% in final
Penicillin/Steptomycin	Hyclone	SV30010	1×100 stocking
0.25% trypsin 0.03% EDTA solution	Corning	25-053-CI
Sodium pyruvate	Corning	25-000-CI	1.0 µM in final
Glucose	Sigma	CAS 50-99-7	25 mM in final
Oligomycin	Sigma	O4876	1.0 µM in final
Carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)	Sigma	C2920	0.5 µM in final
Rotenone	Sigma	R8875	1.0 µM in final
Antimycin A	Sigma	CAS 1397-94-0	1.0 µM in final
Plate wash station	Seahorse Bioscience
Extracellular flux bioanayzer	Seahorse Bioscience	XFe96
Extracellular flux cell culture plate	Seahorse Bioscience	102416-100
Extracellular flux sensor cartridge	Seahorse Bioscience	102416-100
Extracellular flux calibrant solution	Seahorse Bioscience	100840-000
Extracellular flux assay medium	Seahorse Bioscience	102365-100	PH buffered prior to assay