Murine chondrocytes were treated with IL-1β (10 ng/mL) for 24 h in the presence of absence of SC-514 (10 μM). Mitochondrial biogenesis was measured using colorimetric Mitobiogenesis In-Cell ELISA kit (Ab110217, Abcam, Cambridge, MA, USA). Colorimetric assay was performed in 96-well plate format.
Mitobiogenesis in cell elisa kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The MitoBiogenesis™ In-Cell ELISA Kit is a quantitative assay that measures the levels of mitochondrial proteins in cultured cells. The kit provides a simple and efficient method to assess mitochondrial biogenesis without the need for cell lysis or protein extraction.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Qpcrbio sygreen mix separate rox, by PCR Biosystems (1 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
Spectramax m2 spectrophotometer, by Molecular Devices (1 mentions)
Fluostar plate reader, by BMG Labtech (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Mtt dye, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ampkα1 2, by Cell Signaling Technology (1 mentions)
P ampkα1 2, by Santa Cruz Biotechnology (1 mentions)
Antibodies against c myc, by Cell Signaling Technology (1 mentions)
P drp1, by Cell Signaling Technology (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
11 protocols using mitobiogenesis in cell elisa kit
1
Mitochondrial Biogenesis in Chondrocytes
2
Mitochondrial Function Assessment Protocol
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Reagents used in this study include Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA); fetal bovine serum (FBS) (Biosera, Nuaillé, France); idebenone, L-buthionine sulfoximine (BSO), and aconitase assay kit (Cayman Chemical, Ann Arbor, MI, USA); adenosine, penicillin–streptomycin, and mitochondrial membrane potential (MMP) kit (Sigma-Aldrich, St. Louis, MO, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Alfa Aesar, Haverhill, MA, USA); Mitochondrial ToxGloTM assay kit (Promega Corporation, Madison, WI, USA); MitoBiogenesis™ In-Cell ELISA Kit (Abcam, Cambridge, UK); TRIzol® reagent (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA); RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA); qPCRBIO SyGreen Mix Separate-ROX (PCR Biosystems, London, UK); and OmniPur® water (Merck Millipore, Darmstadt, Germany).
3
Mitobiogenesis Quantification via ELISA
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After mitochondrial augmentation, cells were collected and washed twice with PBS. Mitobiogenesis in-Cell ELISA kit (Abcam., Cambridge, UK cat#ab110217) was used to assess protein levels of mitochondrially encoded cytochrome c oxidase subunit 1 (COX-1), normalized to Janus green staining quantification of total cell number, using infinite pro 200 plate reader (TECAN, Männedorf, Switzerland).
4
Mitochondrial Protein Analysis in Bleomycin-Induced Lung Cells
5
Mitochondrial Biogenesis Assay in PC-3 Cells
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PC-3 cells were treated with test compounds at increasing concentrations for 5 days and analyzed as previously described with a colorimetric MitoBiogenesis In-Cell ELISA kit (Abcam, Cambridge, United Kingdom) (12 (link)). Briefly, PC-3 cells were plated in 96-well plates at a density of 2.5 × 103 cells/well in Ham’s F-12K complete medium. After 5 days of incubation with drug, the cellular levels of the mitochondrial DNA-encoded protein, COX1, and the nuclear DNA-encoded protein, SDH-A, were analyzed using the assay kit according to the manufacturer’s protocol. Colorimetric analysis was performed using a SpectraMax M2 spectrophotometer (Molecular Devices, San Jose, CA). Chloramphenicol and ddC were used as positive controls.
6
Evaluating Mitochondrial Proteins in Cells
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Levels of SDHA and COX-1 (MT-CO1) proteins were evaluated with MitoBiogenesis™ In-Cell ELISA Kit (Abcam) on Fluostar plate reader (OMEGA, BMG LABTECH), according to the manufacture’s protocol, after 5 days of exposure to BZ. The results are shown in the graph as the [%] of the untreated control. The total number of cells was determined with Janus green and calculated from a standard curve. The details of the measurement have been described previously [11 (link), 12 (link)].
7
Quantifying Mitochondrial Biogenesis in HeLa Cells
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HeLa cells cultured on the HCGN platform were treated with MAC for 90 min. The HeLa cells were then fixed with 4% paraformaldehyde. Mitochondrial biogenesis was measured using the colorimetric Mitobiogenesis In‐Cell ELISA kit (ab119217, Abcam, UK), which simultaneously quantifies the levels of mitochondrial DNA‐encoded cytochrome c oxidase I (COX‐1) and nuclear DNA‐encoded succinate dehydrogenase A (SDH‐A) within the HeLa cells. The colorimetric assay was performed in a 96‐well plate format. All experiments were performed according to the manufacturer's instructions, as previously reported.
8
Cryptolepine Hydrate Modulates Mitochondrial Dynamics
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Purified cryptolepine hydrate (≥98% purity by HPLC), MTT dye, 2′,7′-dichlorofluorescin diacetate, and Rhodamine 123 were purchased from Sigma-Aldrich (St. Louis, MO). The MitoBiogenesis In-cell ELISA kit was purchased from Abcam (Cambridge, MA). Antibodies against c-Myc, p-Drp1, LKB1, and AMPKα1/2 were purchased from Cell Signaling Technology (Beverly MA). The Opa1 antibody was obtained from Thermo Scientific (Rockford, IL). Antibodies against PGC-1α, Mitofusin 1, Mitofusin 2, Drp1, p-AMPKα1/2, SIRT1, and β-actin and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The ATP Determination kit, MitoTracker Red CMXRos, AlexaFluore 488- and 594-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).
9
Mitochondrial Protein Analysis in Bleomycin-Induced Lung Cells
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Human SAECs, primary (AECIIs) isolated from the mouse lungs and MLE-12
were seeded in 96-well microplates containing 100 μL of culture medium.
Cells were first exposed to bleomycin (10mU/ml) diluted in PBS or vehicle
control (PBS) for 4 hours and then were treated with T3 (15ng/ml) or vehicle
control for 8 hours. The levels of two mitochondrial proteins were measured
simultaneously in each well by using a colorimetric ELISA-kit, according to
manufacturer’s protocol (MitoBiogenesis™ In-Cell ELISA Kit-
Abcam-ab110217)17 (link). The
two proteins are each subunits of a different oxidative phosphorylation enzyme
complex, one protein being subunit I of Complex IV (Cytochrome c oxidase
subunit-COX-IV), which is mitochondrial-DNA-encoded, and Succinate Dehydrogenase
Complex Flavoprotein subunit A-SDHA) which is a 70kDa subunit of Complex II and
nuclear-DNA-encoded.
were seeded in 96-well microplates containing 100 μL of culture medium.
Cells were first exposed to bleomycin (10mU/ml) diluted in PBS or vehicle
control (PBS) for 4 hours and then were treated with T3 (15ng/ml) or vehicle
control for 8 hours. The levels of two mitochondrial proteins were measured
simultaneously in each well by using a colorimetric ELISA-kit, according to
manufacturer’s protocol (MitoBiogenesis™ In-Cell ELISA Kit-
Abcam-ab110217)17 (link). The
two proteins are each subunits of a different oxidative phosphorylation enzyme
complex, one protein being subunit I of Complex IV (Cytochrome c oxidase
subunit-COX-IV), which is mitochondrial-DNA-encoded, and Succinate Dehydrogenase
Complex Flavoprotein subunit A-SDHA) which is a 70kDa subunit of Complex II and
nuclear-DNA-encoded.
10
Mitochondrial Biogenesis Quantification
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MitoBiogenesisTM In-Cell ELISA kit purchased from abcam (#ab140359) for measuring mitochondrial biogenesis was used in SH-SY5Y cells. Two proteins are detected following the manufacturer’s instructions; COXI is mtDNA-encoded protein and SDH-A is nDNA-encoded protein. The COXI protein synthesis relative to SDH-A synthesis is the quantitative measurement for mitochondrial biogenesis and content.
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