The mitochondrial protein fractions were prepared using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific, USA).
Mitochondrial isolation kit for cultured cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mitochondrial Isolation Kit for Cultured Cells is a laboratory tool designed to isolate mitochondria from cultured cells. It provides a standardized procedure for the extraction and purification of mitochondria, a key organelle within eukaryotic cells.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Phosphorylated bcl 2, by Cell Signaling Technology (1 mentions)
Phosphorylated c jun ser73, by Cell Signaling Technology (1 mentions)
Total c jun, by Cell Signaling Technology (1 mentions)
Phosphorylated mtor ser2448, by Cell Signaling Technology (1 mentions)
5 bromo 2 deoxyuridine, by Roche (1 mentions)
Anti bromodeoxyuridine, by Roche (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Total mtor, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Mitochondria isolation kit for tissue, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Mak193, by Merck Group (1 mentions)
Mak206, by Merck Group (1 mentions)
Cytochrome oxidase activity colorimetric assay kit, by Abcam (1 mentions)
Proteinpilot software, by AB Sciex (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
29 protocols using mitochondrial isolation kit for cultured cells
1
Mitochondrial Protein Fractionation
2
Autophagy Regulation in Cell Signaling
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GPD was obtained from the Ambo Institute (Seoul, Republic of Korea). The antibody against LC3 was purchased from ABGENT Inc. (San Diego, CA), while antibodies against phosphorylated AMPKα (Thr172), total AMPKα, phosphorylated c-Jun NH2-terminal kinases (JNKs; Thr183/Tyr185), total JNK, Beclin-1, phosphorylated Bcl-2, cytochrome C, PARP, caspase-3, 7, 9, phosphorylated ATF2, ATF, phosphorylated c-Jun (Ser73), total c-Jun, phosphorylated mTOR (Ser2448) and total mTOR were purchased from Cell Signaling Technology (Danvers, MA). The antibodies against p62/SQSTM1, Bcl-2, α-tubulin and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The BCA protein assay kit and mitochondrial isolation kit for cultured cells were obtained from Thermo Scientific (Rockford, IL). Puromycin was purchased from InvivoGen (San Diego, CA). 5-bromo-2′-deoxyuridine and anti-bromodeoxyuridine were purchased from Roche Applied Science (Indianapolis, IN).
3
Isolation and Characterization of Mitochondria
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Mitochondrial organelles were isolated from 2 × 107 cells treated with iPA 10 µM for 24 h using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Monza, Italy, COD# 89874). Mitochondrial preparations were obtained following the manufacturer’s instructions and resolved by SDS-PAGE electrophoresis.
4
Isolating Mitochondrial Fractions
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Fresh tubules and cultured cells were washed with cold PBS first. Mitochondrial fractions were isolated from tubules using Mitochondria Isolation Kit for Tissue and from TECs or PTCs using Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific) according to the manufacturer’s protocols.
5
Mitochondrial Fractionation and Localization
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Mitochondrial fractionation was performed using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific, MA, USA) following the manufacturer’s instructions. In brief, 1 × 107 cells were collected and incubated in mitochondria isolation buffers on ice for 5 min. After homogenization, nuclei and intact cells were discarded after centrifugation at 700 g for 10 min at 4°C. The supernatant enriched with the cytosolic fraction and the pellet enriched with the mitochondrial fraction were obtained after centrifugation at 12,000 g for 15 min at 4°C. For mitochondrial protein-localization assays as previously described [25 (link)], the purified mitochondria were divided into three groups. The first group was not treated, as a control. The second group was treated with 3 μM proteinase K (Invitrogen, MA, USA). The third group was treated with 3 μM proteinase K and 0.1% Triton X-100 solution. Three groups of samples were placed in a 37°C water bath for 30 min. All samples were prepared for further immunoblotting.
6
Mitochondrial Isolation from Cultured Cells
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Cells grown in 60-mm dishes were subjected to mitochondrial purification using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific). Briefly, the attached cells (1 × 107) were incubated with 800 μL reagent A on ice for 2 minutes. Then, cell lysates were transferred to Eppendorf tube, followed by treatment with 10 μL reagent B on ice and the reactions were vortexed every minute at maximum speed for a total of 5 minutes. Reagent C (800 μL) was successively added to the reactions, which then were centrifuged at 700 × g, 4ºC for 10 minutes. Supernatant was saved and continuously centrifuged at 12,000 × g, 4ºC for 15 minutes. The resulting supernatant is the cytosolic fraction. The pellet (mitochondrial fraction) was washed with 500 μL reagent C at 12,000 × g, 4ºC for 5 minutes. After discarding the supernatant, the pelleted mitochondria are active and were used in the following study of mitochondrial bioactivity. Alternatively, the mitochondria were lyzed with RIPA lysis buffer for subsequent Western blot analysis.
7
Mitochondria Isolation and Lysis
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Cells were counted, and mitochondria isolated from an equal number of cells using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific, 89874) following the manufacturer's instructions. Centrifugation was performed at 3000 g for 15 min to obtain a more purified mitochondrial fraction. Isolated mitochondria were lysed in equal volumes of 2% CHAPS in TBS. The cytoplasmic fraction was also retained.
8
Mitochondrial Isolation and Purity Verification
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After treatment, the mitochondria were fractionated using a mitochondrial isolation kit for cultured cells (1 × 107) (Thermo Fisher Scientific) according to the manufacturer's instructions. To ensure the purity of the fraction, COXIV and GAPDH were respectively used as mitochondrial and cytosolic markers.
9
Mitochondrial Isolation and Transfer in MSC-MDM Co-culture
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Mitochondrial isolation from MSC was performed using the mitochondrial isolation kit for cultured cells (Thermo Fisher Scientific (Paisley, UK, www.thermofisher.com )) according to manufacturer's instructions. Isolated mitochondria were resuspended in RPMI 1% FCS according to the final cell count of MSC, maintained on ice and used immediately for artificial transfer. Transfer of isolated mitochondria to MDM in vitro was performed according to Caicedo et al. 30 .
10
Mitochondrial Enzyme Activity Assays
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Enzymatic activity assays were conducted using the mitochondrial fraction following the manufacturer’s instructions for fumarase activity colorimetric assay kit MAK206 (Sigma-Aldrich), Aconitase activity assay kit MAK051 (Sigma-Aldrich), and citrate synthase activity kit MAK193 (Sigma-Aldrich). The assays were carried out with isolated mitochondrial protein using mitochondrial isolation kit for cultured cells (Thermo Fisher Scientific, Inc. #89874, Pierce, Rockford, Waltham, MA) following the manufacturer’s recommendations. Once the mitochondrial protein was obtained, it was quantified using BCA assay (standards prepared in Chaps buffer 2% in 1 × TBS). The amount of protein was equalized to 1 μg/μl in assay buffer for each activity. A total of 25–30 μg of mitochondrial protein were analyzed in each activity assay with replicates, aconitase and citrate synthase activity was expressed in nmol/min/μL (milliunits/μL-mU/μL) and fumarase nmol/min/mL (milliunits/mL- mU/mL).
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