For mitochondrial uptake, MDA-MB-468 cells (20 × 106) were treated with 1 or 2μM of AuPhos-19 and incubated for 18 h at 37°C. The medium was removed, and the cells were washed with PBS solution (1 mL × 3), trypsinized, and centrifuged. Mitochondria extraction from the cells was then done using the mitochondria extraction kit (ThermoFisher Scientific) according to the manufacturer’s protocol. The separated mitochondria pellets were mineralized with 70% HNO3 followed by dilution with DI water as needed. Analysis of gold content was done by GF-AAS and cellular gold levels were expressed as pmol of Au per million cells.
Mitochondria extraction kit
Manufactured by Thermo Fisher Scientific
The Mitochondria Extraction Kit is a laboratory tool designed to isolate and extract mitochondria from cells or tissues. It provides a standardized protocol for the separation and purification of these cellular organelles, which are essential for energy production within the cell.
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Lab products found in correlation
Control igg antibody, by Cell Signaling Technology (1 mentions)
Protein a g sepharose, by GE Healthcare (1 mentions)
Sds page sample buffer, by Beyotime (1 mentions)
Nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Lysosome extraction kit, by Merck Group (1 mentions)
6 protocols using mitochondria extraction kit
1
Mitochondrial Uptake of AuPhos-19
2
Mitochondria Isolation and Protein Analysis
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Neuron cells were cultured in a 6-well plate to 80% confluency. Cytosolic and mitochondria extracts were prepared using a mitochondria extraction kit (Thermo Fisher Scientific). Briefly, cells were resuspended in lysis buffer and disrupted with a Dounce homogenizer. Then, homogenates were centrifuged at 700 ×g to pellet nuclei and cell debris. After centrifuging the supernatants at 12,000 ×g, the cytosolic fractions (supernatant) were collected. Next, the supernatant was further lysed by mitochondria isolation reagent. The mitochondria fractions were obtained after centrifuging at 12,000 ×g and discarding the supernatant. Thereafter, protein concentrations were quantified. Lysates containing 500 μg protein were incubated with target antibodies (anti-TXNIP antibody, anti-TRX1 antibody, and anti-TRX2 antibody) or control IgG antibody (Cell Signaling Technology) at 4°C for 4 h. Then, 50 μL of Protein A/G Sepharose (GE Healthcare) was added, followed by incubation at 4°C overnight. After incubation, beads were washed and resuspended in 50 μL of 2 × SDS-PAGE sample buffer. The sample was heated and collected for western blot analysis.
3
Mitochondrial and Cytosolic Fractionation
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For preparation of cell mitochondrial and cytosolic fractions, we used a mitochondria extraction kit (ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, cells were resuspended in lysis buffer and then disrupted with a Dounce homogenizer. Homogenates were centrifuged at 700 × g for 10 min at 4°C to pellet nuclei and cell debris. Supernatants were subsequently centrifuged at 12,000 × g for 15 min at 4°C, and the cytosolic fractions (supernatants) were collected. Pellets (heavy membranes enriched with mitochondria) were boiled with SDS-PAGE sample buffer (Beyotime) and analyzed by Western blotting. To determine the quality of cytosolic and mitochondrial separation, both fractions were assessed by immunoblotting for the mitochondrial marker cox-IV.
4
Isolation of cardiac cellular fractions
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At the end of 2 hours of postischemic reperfusion, heart tissues were immediately collected for separation of cytosolic and nuclear fractions and for isolation of mitochondria. Hearts were cleared of blood by washing thoroughly in Tyrode buffer and aortic and atrial sections removed from the ventricles. Ventricular tissue was freeze-clamped in liquid nitrogen and stored until fractionated to isolate cytosolic and nuclear fractions (Nuclear and Cytoplasmic Extraction Kit, Thermo, Chicago, IL) and mitochondria (Mitochondria Extraction Kit, Thermo, Chicago, IL) according to the manufacturer's protocol as described [3 (link)].
5
Mitochondrial Isolation and JC-1 Staining
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The mitochondrial isolation from heart tissues was performed according to the manufacturer's instructions as per the Mitochondria Extraction Kit (Thermo Fisher Scientific). The isolated intact mitochondria were incubated with 2 μM JC‐1 stain in black 96‐well microplate for 10 minutes at 37°C. The fluorescent signal was determined by a fluorescence plate reader (Synergy HT BioTek) at excitation/emission of 485/535 nm for green fluorescence and 560/595 nm for red fluorescence.
6
Quantifying Intracellular Ruthenium Distribution
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The co-localisation of the compound was determined by measuring the Ru content inside the cell via ICP-MS. 20 . 10 6 cells were incubated with the compound (20 μM) for 4 h at 37°C in the dark.
After this time, the cells were detached with trypsin and harvested. The number of cells was accurately counted. The amount was equally divided. In the first portion, the nucleus was extracted using a nucleus extraction kit (Thermo Scientific); in the second portion, the mitochondria was extracted using a mitochondria extraction kit (Thermo Scientific); in the third portion, the lysosome was extracted using a lysosome extraction kit (Sigma Aldrich); in the fourth portion, the golgi apparatus was extracted using a golgi apparatus extraction kit (Sigma Aldrich) and in the fifth portion, the endoplasmic reticulum was extracted using a endoplasmic reticulum extraction kit (Sigma Aldrich). Each sample was digested using a 60% HNO3 solution for three days. The solution was evaporated and each sample was diluted to solution of 2% HCl in water. The Ru content was determined using an ICP-MS apparatus and comparing the results with the Ru references. The Ru content was then associated with the number of cells.
After this time, the cells were detached with trypsin and harvested. The number of cells was accurately counted. The amount was equally divided. In the first portion, the nucleus was extracted using a nucleus extraction kit (Thermo Scientific); in the second portion, the mitochondria was extracted using a mitochondria extraction kit (Thermo Scientific); in the third portion, the lysosome was extracted using a lysosome extraction kit (Sigma Aldrich); in the fourth portion, the golgi apparatus was extracted using a golgi apparatus extraction kit (Sigma Aldrich) and in the fifth portion, the endoplasmic reticulum was extracted using a endoplasmic reticulum extraction kit (Sigma Aldrich). Each sample was digested using a 60% HNO3 solution for three days. The solution was evaporated and each sample was diluted to solution of 2% HCl in water. The Ru content was determined using an ICP-MS apparatus and comparing the results with the Ru references. The Ru content was then associated with the number of cells.
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