Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Summary

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Abstract

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

Protocol

High Resolution Clear Native Gel Electophoresis (hrCNE) for Mitochondrial Complex In-Gel Activity Assays

Human embryonic stem cells (hESCs) were maintained on a monolayer of ˠ-irradiated or mitomycin C-treated mouse embryonic fibroblasts (MEFs), then switched to growth on Matrigel for 5 days in MEF-conditioned medium using standard conditions. To remove contaminating MEFs, hESCs were re-plated on Matrigel and grown in MEF-conditioned medium for an additional 3 days. Immunofluorescence microscopy for pluripotency markers Oct-4 and SSEA-4 was used to confirm the lack of hESC differentiation.

1. Wash cells with warm 1x PBS, pH 7....

Discussion

In this video, we have described the extraction of mitochondrial protein complexes from human embryonic stem cells, their separation by high resolution clear native gel electrophoresis (hrCNE), and the subsequent analysis by in-gel catalytic activity assays. hrCNE resolves mitochondrial membrane protein complexes at a resolution comparable to that of blue native gel electrophoresis and is superior for in-gel activity assays. This technique can be employed not only to quantify mitochondrial respiratory complexes I-V but a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5M EDTA	Fisher Scientific	BP120-1	adjust pH to 8.0 with NaOH
1M NaCl	Sigma-Aldrich	S3014-500MG
1M Sodium succinate	Fisher Scientific	S413-500
0.1M Sodium phosphate buffer pH 7.2	Fisher Scientific	BP332-1 and BP329-1	Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C
1M Tricine, pH 7.0 (adjusted with NaOH)	EMD Millipore	9010	store at 4 C
1M imidazole/HCl, pH 7.0	Sigma-Aldrich	I-2399	store at 4 C
1M Tris-HCl, pH 7.4	Fisher Scientific	BP153-1
20% (wt/vol) dodecyl-b-D-maltoside (DDM)	Fluka	44205-500MG	Dissolved in water. Store 1 ml aliquots at –20 C.
10% (wt/vol) digitonin (>50% purity, used without recrystallization)	Fluka	37008-1G	Dissolved in water. Store 0.1–1 ml aliquots at –20 C. Should be warmed up to 95 C before use.
2M 6-aminohexanoic acid	Fluka	07260-100G	store at 4 C
10x Ponceau S/glycerol stock solution	Sigma-Aldrich	P-3504 and G6279-1L	0.1% Ponceau S, 50% glycerol, (wt/vol) in water.
10% w/v sodium deoxycholate	Fluka	30970-25G	Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.
250 mM phenazine methasulfate (PMS)	TCI America	P0083	Dissolved in DMSO and aliquoted. Store at –20 C.
Nitro-blue tetrazolium (NBT)	Amresco	0329-1G
3,3’-diaminobenzidine tetrahydrochloride (DAB)	TCI America	D0078
Pb(NO3)2	Fisher Scientific	L62-100
MgSO4	Fisher Scientific	BP213-1
Tris base	Fisher Scientific	BP152-10
Glycine	Fisher Scientific	G45-212
Adenosine 5’-triphosphate disodium salt (ATP)	Sigma-Aldrich	A2383-5G	Grade I, minimum 99%
beta-nicotineamide adenine dinucleotide (NADH) reduced form	Sigma-Aldrich	N-8129
Cytochrome c From horse heart	Sigma-Aldrich	C2506-250MG
Sucrose	Fisher Scientific	BP220-212
100x protease inhibitor cocktail	Sigma-Aldrich	P8340	Store at – 20 C.
Dimethyl sulfoxide (DMSO)	Fisher Scientific	D128-500
Trypan blue Stain 0.4%	GIBCO, by Life Technologies	15250-061
10x Trypsin (0.5%)	GIBCO, by Life Technologies	15400-054
Trypsin Inhibitor	GIBCO, by Life Technologies	17075-029