Mitochondria isolation kit for culture cells

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Mitochondria Isolation Kit for Culture Cells is a laboratory tool designed to extract and purify mitochondria from cultured cells. It provides a simple and efficient method to obtain intact mitochondria for further analysis and experimentation.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Nativepage bis tris gel, by Thermo Fisher Scientific (1 mentions) Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Anti bax, by Cell Signaling Technology (1 mentions) Anti bcl 2, by Cell Signaling Technology (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Cell culture medium, by Thermo Fisher Scientific (1 mentions) Hyperfect transfection reagent, by Qiagen (1 mentions) Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (1 mentions) Anti foxo3a, by Cell Signaling Technology (1 mentions) Anti cytochrome c cyt c, by Cell Signaling Technology (1 mentions) Anti histone h3, by Cell Signaling Technology (1 mentions) Anti bim, by Cell Signaling Technology (1 mentions) Anti caspase 9, by Cell Signaling Technology (1 mentions) Anti caspase 3, by Cell Signaling Technology (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Mitochondrial membrane potential assay kit with jc 1, by Keygen Biotech (1 mentions)

Spelling variants of this product

Mitochondria isolation kit (295 citations) Cell mitochondria isolation kit (2 citations)

9 protocols using mitochondria isolation kit for culture cells

Mitochondria Isolation from HEK293T-FcγRII Cells

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Mitochondria were isolated from HEK293T-FcγRII cells using Mitochondria Isolation Kit for Culture Cells (Thermo Fisher) with a detergent-free homogenization method (option B in the manufacturer’s instructions). Instead of using a dounce homogenizer, cells were ruptured by 15 strokes of a 26-gauge needle. Equivalent amounts of postnuclear supernatant (PNS) and cytoplasmic and mitochondrial fractions were analyzed with immunoblotting.

Kubori T., Lee J., Kim H., Yamazaki K., Nishikawa M., Kitao T., Oh B.H, & Nagai H. (2022). Reversible modification of mitochondrial ADP/ATP translocases by paired Legionella effector proteins. Proceedings of the National Academy of Sciences of the United States of America, 119(23), e2122872119.

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Isolation and Analysis of Cardiac Mitochondria

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Cardiac mitochondria were isolated using differential centrifugation as described previously [20 (link)]. In brief, heart from 3–4-month-old mouse was excised, washed in ice-cold Chappell-Perry buffer, homogenized with a polytron homogenizer and subjected to two-step differential centrifugation (500 g and 3000 g). Mitochondrial pellet from 3000 g centrifugation was washed and subjected to further experiments. Mitochondria from cultured cells were isolated using the Mitochondria Isolation Kit for Culture Cells (ThermoFisher Scientific, 89874). To assess the integrity of mitochondrial respiration complexes, mitochondrial protein was solubilized with 1% DDM and separated in 4–16% NativePAGE™ Bis-Tris Gel (ThermoFisher Scientific, BN2005, BN1002BOX).

Zhang R., Shen Y., Zhou L., Sangwung P., Fujioka H., Zhang L, & Liao X. (2017). Short-term administration of Nicotinamide Mononucleotide preserves cardiac mitochondrial homeostasis and prevents heart failure. Journal of molecular and cellular cardiology, 112, 64-73.

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Isolation of Mitochondria from Myotubes

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Mitochondria were isolated from myotubes as instructed (Mitochondria Isolation Kit for Culture Cells, Thermo Scientific, 89874). Briefly, 2 × 10⁷ cell pellet was resuspended with mitochondria isolation reagent A and vortexed at medium speed for 5 s and then incubated on ice for 2 min. After incubation, 10 µl of reagent B was added and vortexed at maximum speed for 5 s followed by an additional incubation on ice for5min. After adding 800 µl of reagent C, tubes were inverted and then centrifuged (700g for 10min at 4°C). In a new tube, the supernatant was centrifuged again (12,000g for 15min at 4°C). The mitochondrial pellet was spun (12,000g for 5 min) and cleaned again by adding 500 µl of reagent C before mitochondrial lysis using RIPA buffer.

Ribas V., Drew B.G., Zhou Z., Phun J., Kalajian N.Y., Soleymani T., Daraei P., Widjaja K., Wanagat J., de Aguiar Vallim T.Q., Fluitt A.H., Bensinger S., Le T., Radu C., Whitelegge J.P., Beaven S.W., Tontonoz P., Lusis A.J., Parks B.W., Vergnes L., Reue K., Singh H., Bopassa J.C., Toro L., Stefani E., Watt M.J., Schenk S., Akerstrom T., Kelly M., Pedersen B.K., Hewitt S.C., Korach K.S, & Hevener A.L. (2016). Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females. Science translational medicine, 8(334), 334ra54.

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Apoptosis Signaling Pathway Analysis

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Cell culture medium (Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F-12)), fetal calf serum, bovine serum albumin (BSA), and protease inhibitor cocktail were obtained from GIBCO/Invitrogen (Carlsbad, CA, USA). Anti-SGK1 and anti-phospho-SGK1 (Ser422) were purchased from Merck (Darmstadt, Germany). An anti-ClC-3 antibody was obtained from Alomone Labs (Jerusalem, Israel). Anti-Bcl2, anti-Bax, anti-caspase-9, anti-cleaved caspase-9, anti-caspase-3, anti-cleaved caspase-3, anti-cytochrome c (Cyt-c), anti-Bim, anti-FOXO3a, anti-phospho-FOXO3a (Ser316), anti-poly ADP-ribose polymerase (PARP), anti-cleaved PARP, anti-histone H3 and anti-Cyt-c oxidase subunit IV (COX IV) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Hyperfect transfection reagent was purchased from Qiagen (Valencia, CA, USA). The Cell Counting Assay Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (MD, Japan). H₂O₂ was purchased from Merck. The Annexin V-PE/7-AAD Apoptosis Detection Kit and the Mitochondrial Membrane Potential Assay Kit with JC-1 were purchased from Keygen Biotech (Nanjing, China). The Mitochondria Isolation Kit for culture cells was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Unless otherwise indicated, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Chen B.Y., Huang C.C., Lv X.F., Zheng H.Q., Zhang Y.J., Sun L., Wang G.L., Ma M.M, & Guan Y.Y. (2020). SGK1 mediates the hypotonic protective effect against H2O2-induced apoptosis of rat basilar artery smooth muscle cells by inhibiting the FOXO3a/Bim signaling pathway. Acta Pharmacologica Sinica, 41(8), 1073-1084.

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Mitochondrial Enzyme Activities Assay

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Enzymatic activities of complexes I, II, III and IV were measured in mitochondria from all groups of cells (untreated N2a, N2a cells treated with Mdivi-1, Drp1 RNA silenced in N2a cells, N2a cells transfected with full-length Drp1 and treated with Mdivi-1 and N2a cells transfected with full-length Drp1). We used Mitochondria Isolation Kit for Culture Cells (ThermoFisher Scientific Cat# 89874, 1680 Campus Delivery, Fort Collins, Colorado, USA) to isolate intact mitochondria.

Manczak M., Kandimalla R., Yin X, & Reddy P.H. (2018). Mitochondrial division inhibitor 1 reduces dynamin-related protein 1 and mitochondrial fission activity. Human Molecular Genetics, 28(2), 177-199.

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Mitochondrial Isolation and Membrane Fractionation

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Mitochondrial isolation was performed using the Mitochondria Isolation Kit for Culture Cells (Thermo Fisher Scientific, Rockford, IL, USA) following the suggested protocol. Membrane fractionation was performed using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) according to the suggested protocol. Cells were treated with 5.03 μM TP4 in membrane fractionation experiments. For the co-immunoprecipitation (coIP) experiment, equal amounts of protein lysate (500 μg) form PCDHB13-tGFP- and control tGFP-overexpressing A549 cells were harvested for IP using anti-tGFP magnetic beads (OriGene Technologies), in accordance with the recommended protocol. Cell extract preparation and Western blot were performed as previously described [14 (link)]. Protein samples were loaded in duplicate gels and transferred to separate membranes for probing with different antibodies. The results were expressed as relative densitometric units (RDU; densitometric intensities of FOSB+FOSΔB divided by that of GAPDH). Complete and unedited Western blot images were shown in Figure S9.

Ting C.H., Lee K.Y., Wu S.M., Feng P.H., Chan Y.F., Chen Y.C, & Chen J.Y. (2019). FOSB–PCDHB13 Axis Disrupts the Microtubule Network in Non-Small Cell Lung Cancer. Cancers, 11(1), 107.

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GPR120 Activation and Mitochondrial Function

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Grifolic acid and TUG891 were obtained from R&D Inc. (Minnneapolis, USA). GW9508, EPA, GPR120 polyclonal antibody, MTT and Cellular ATP assay kits were bought from Sigma-Aldrich (St. Louis, USA). Annexin V-FITC/PI staining kits were the products of BD Pharmingen (San Jose, USA). Rat GPR120 siRNA, lipofectamine RANiMAX, DMEM, FBS, JC-1 and Mitochondria Isolation Kit for Culture Cells were obtained from Thermo Fisher Scientific (Waltham, USA). NAD/NADH Assay Kits were the products of Abcam (Cambridge, UK). Protein extraction kits were bought from Bio-Rad (Hercules, USA). RNA isolation kits, reverse transcription kits and PCR kits were the products of Takara Biotechnology (Dalian, China).

Zhao Y., Zhang L., Yan A., Chen D., Xie R., Liu Y., Liang X., Zhao Y., Wei L., Yu J., Xu X, & Su X. (2018). Grifolic acid induces GH3 adenoma cell death by inhibiting ATP production through a GPR120-independent mechanism. BMC Pharmacology & Toxicology, 19, 26.

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Mitochondrial DNA Purification Protocol

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mtDNA was purified by adapting a previously developed protocol (18 (link)). About 20 million cells were first lysed by douncing with a 2 ml tissue mortar/pestle set (Fisher Scientific K885300-0002). Intact mitochondria were then purified according to manufacturer’s instructions using the Mitochondria Isolation Kit for Culture Cells (Thermofisher 89874). The intact mitochondria were further treated with 400U DNase I (Sigma D5025) in 200 μl modified DNase I buffer (5 mM HEPES buffer pH 7.4, 210 mM mannitol, 70 mM sucrose, 10 mM MgCl₂, 5 mM CaCl₂, 0.2% bovine serum albumin and 1× Complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail) for 30 min at 37°C to digest away any DNA outside of mitochondria. Mitochondria were then centrifuged at 12 000 g for 5 min at 4°C. The mitochondrial pellet was washed with a 1:1 mixture of reagent A plus reagent C from the kit, re-centrifuged at 12 000 g for 5 min at 4°C, decanted then frozen in liquid nitrogen and stored at −80°C before DNA extraction.

Koh C.W., Goh Y.T., Toh J.D., Neo S.P., Ng S.B., Gunaratne J., Gao Y.G., Quake S.R., Burkholder W.F, & Goh W.S. (2018). Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome. Nucleic Acids Research, 46(22), 11659-11670.

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Quantifying Cellular ATP Levels

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ATP measurements were performed using the Enliten^® ATP assay system (Promega). Briefly, triplicate cells were split into two aliquots. One aliquot was subjected to mitochondria isolation using a mitochondria isolation kit for culture cells (ThermoFisher Scientific). Both the intact cells and isolated mitochondria pellets were resuspended in saline, with 5% taken out for SDS-PAGE analysis, the remaining saline suspension was treated 1:1 with 5% TCA (trichloroacetic acid), vortexed, then neutralized with half the original volume of 1 M Tris pH 7.75. The sample was diluted 1:1 with 1 M Tris pH 7.75. 10 μL of the diluted sample was added to 90 μL of the ATP assay reaction mix and plated in duplicate on a white-bottomed 96-well plate. Luminescence was then measured. The 5% of the original saline sample was diluted with 5× SDS buffer and run on a 7.5% SDS-PAGE gel and probed for SDHA (abcam, ab14715). The luminescence measurement was then normalized to the amount of SDHA.

Green N.H., Galvan D.L., Badal S.S., Chang B.H., LeBleu V.S., Long J., Jonasch E, & Danesh F.R. (2019). MTHFD2 links RNA methylation to metabolic reprogramming in renal cell carcinoma. Oncogene, 38(34), 6211-6225.

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