After the indicated treatments, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% NP-40) supplemented with protease inhibitors cocktail (Merck Millipore, Darmstadt, Germany) to prepare the whole-cell lysates. Moreover, cells subjected to designated treatments were collected and pooled together for lysosome isolation. Lysosomal fractions were extracted from cell homogenates by differential centrifugation followed by density centrifugation according to the manufacturer’s protocol (Lysosome Extraction Kit; Sigma-Aldrich; LYSISO1). Briefly, cell homogenates were centrifuged at 1000 × g for 10 min at 4 °C, then the supernatant fraction was centrifuged at 20 000 × g for 20 min at 4 °C. The resulting supernatant fraction was collected as cytosolic fraction and the pellet contains lysosomes and other organelles (crude lysosomal fraction). The pellet fractions were subjected to additional centrifugation (150 000 × g for 4 h at 4 °C). The final pellet (lysosomal) fraction was lysed in the lysis buffer described in the procedure for western blotting.
Lysosome extraction kit
Manufactured by Merck Group
Sourced in United States
The Lysosome Extraction Kit is a laboratory tool used to isolate and purify lysosomes, which are organelles found within cells. The kit provides a standardized method for the effective separation and extraction of lysosomes from cell samples, enabling researchers to study their structure, function, and composition.
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6 protocols using lysosome extraction kit
1
Lysosome Isolation and Enrichment
2
Lysosomal Ca2+ Dynamics Monitoring
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The cells in each group were washed three times with PBS, and the intracellular lysosome was extracted by lysosome extraction kit (Sigma-Aldrich, St. Louis, MO, USA). Then, 3 µmol/L Fluo-3AM solution was immediately added, and incubated in a incubator at 37°C for 30 min. After aspirating the working fluid, lysosome was rinsed with PBS for three times, and PBS was added to balance for 10 min, and finally for observation. By using Ca2+ analyzer, the image and time was obtained with real-time monitoring via NIS-Element AR3.0 (Nikon) software system, with a period of 6 min. Lysosomal Ca2+ intensity was expressed as R340/380 (Wavelengths at 340 and 380 nm, respectively). Each experiment was detected at least 10 cells, baseline was also detected (baseline fluctuates within a relatively small range), with each experiment repeating at least three times.
3
Lysosome Isolation and Purification Protocol
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All the steps were performed according to the manufacturer’s protocol at 4 °C (Lysosome Extraction Kit, Sigma, LYSISO1). Briefly, adherent cells were trypsinized and washed with cold PBS. The cell pellet was resuspended and lysed in a 7-ml Dounce homogenizer. Then, the cell homogenates were centrifuged for 10 min at 1000 × g. Subsequently, the supernatants were centrifuged for 20 min at 20,000 × g to pellet the lysosomes and other organelles. Following density gradient centrifugation for 4 h at 150,000 × g in an SW50.1 rotor, the highest (least dense) band was removed and diluted in PBS. The lysosomes were washed and pelleted by centrifugation at 20,000 × g for 20 min. Finally, the precipitate was analyzed by western blotting or immunofluorescence staining.
4
Isolation of Lysosomal Fractions from Cells
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Lysosomal fractions were extracted from cell homogenates by Lysosome Extraction Kit (Sigma-Aldrich; LYSISO1) according to the manufacturer’s protocol. Briefly, cell homogenates were centrifuged for 10 min at 1000 × g at 4 °C. The supernatant fraction was centrifuged for 20 min at 20,000 × g at 4 °C to pellet lysosomes and other organelles, and the resulting supernatant fraction was collected as cytosolic fraction. The pellet fractions were subjected to additional centrifugation. The final pellet (lysosomal) fraction was lysed in the lysis buffer described in the procedure. Samples were subjected to immunoblotting.
5
Isolation and Extraction of Lysosomal Fractions
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Lysosomes were isolated emplyoing a lysosome isolation kit (Abcam, #ab234047) following the manufacturer's protocol. Briefly, cells were collected into 15 mL conical tubes and isolated using an isolation buffer. The supernatant was centrifuged at 500 ×g for 10 min at 4°C and layered onto a discontinuous density gradient. Lysosomes were further isolated via ultracentrifugation (Beckman, Optima XE-100) at 145,000 g for 2 h at 4°C. Lysosomal fractions were extracted from cell homogenates using a lysosome extraction kit (Sigma-Aldrich; LYSISO1). Cell homogenates were centrifuged at 1000 × g for 10 min at 4°C. The supernatant fraction was centrifuged at 20,000 × g for 20 min at 4°C to pellet lysosomes and other organelles. The supernatant was collected as the cytosolic fraction. Pellet fractions were subjected to additional centrifugation. The ultimate pellet, lysosomal fraction, was lysed using the lysis buffer described within the procedure. Samples were then subjected to western blot analysis.
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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