A549 cells were collected after incubation for 2 h by scraping with the culture medium and rinsing two times with Hank's balanced salt solution. Isolation of the lysosome-rich fraction from A549 cells was performed with Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo Scientific, Rockford, IL). After A549 cells had been added to 250 μM of Lysosome Enrichment Reagent A, the mixture was mixed by Vortex for 5 s, incubated on ice for 2 min, and treated by mild sonication (6–9 W of power) for 10 s. The suspension was mixed with 250 μM of Lysosome Enrichment Reagent B and centrifuged at 500×g for 10 min at 4 ℃. The supernatant was overlayed on the top of a prepared discontinuous density gradient and gradient centrifuged at 145,000×g for 2 h at 4 ℃. The lysosome-rich band was mixed with five volumes of phosphate buffered saline (PBS) and rinsed by centrifugation at 18,000×g for 30 min. The residue was rinsed with PBS in a similar manner.
Lysosome enrichment kit for tissue and cultured cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lysosome Enrichment Kit for Tissue and Cultured Cells is a tool designed to isolate and enrich lysosomes from various biological samples, including tissues and cultured cells. The kit provides a standardized protocol and reagents to facilitate the purification of these organelles, which play a crucial role in cellular processes such as degradation and recycling.
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Lab products found in correlation
Beckman ultracentrifuge, by Beckman Coulter (1 mentions)
Chaps, by Merck Group (1 mentions)
Tris buffered saline (tbs), by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
Chaps detergent, by Thermo Fisher Scientific (1 mentions)
7 protocols using lysosome enrichment kit for tissue and cultured cells
1
Lysosome Enrichment from A549 Cells
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Lysosome Enrichment and Protein Analysis
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Isolation and Enrichment of Lysosomes
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Lysosomes isolation was carried out with the Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo Scientific), according to the manufacturer’s protocol77 . Briefly, ~ 25 million differentiated neurons (pellet sizes 80–100 mg) were used for each 8h-compound treatment and purification, with n = 2 biological replicates for each cell line. Lysis buffers were supplemented with Halt protease and phosphatase inhibitors cocktail (Thermo Scientific). After lysis in Lysosome Enrichment Reagent A + B, samples were centrifugated at 500 × g for 10 min and an aliquot of the supernatant was kept as a sample of the cytosolic fraction (total protein minus nuclei). The remaining lysate was mixed with OptiPrep Cell Separation Media and overlaid onto an OptiPrep density gradient. Samples were ultra-centrifugated at 145,000 × g for 2 h at 4 °C, and the lysosome fractions (top gradient band) were transferred to eppendorf tubes and kept on ice. Lysosomes were washed and pelleted at 18,000 ×g for 30 min at 4 °C, and then lysed in 2% (w/v) CHAPS detergent in Tris-buffered saline (Thermo Scientific), supplemented with Halt protease and hosphatase inhibitors cocktail (Thermo Scientific), and centrifugated at 18,000 × g at 4 °C for 5 min. The clarified supernatants containing lysosomal proteins were analyzed by SDS-PAGE western blot (Supplementary Table 2 : antibodies and dilutions).
4
Lysosomal Fractionation from Cultured Cells
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Purification of lysosomal fractions from cultured cells was performed using the Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo Fisher Scientific, Waltham, MA) following the manufacturer instructions. Briefly, 50 mg of pelleted cells were homogenized in Lysosome Enrichment Reagent A using a dounce homogenizer (30 strokes), followed by the addition of the same volume of Lysosome Enrichment Reagent B. The nuclei, cell debris, and mitochondria were removed by a 10-min centrifugation at 1,000 × g at 4 °C. A “crude lysosomal fraction (CLF)” containing the lysosomes, mitochondria, peroxisomes, endoplasmic reticulum and microsomes was obtained by centrifugation of supernatants at 20,000 × g for 20 min at 4 °C. Lysosomes were purified from the CLF by the ultracentrifugation (150,000 × g for 4 h in a Beckman SW 60 Ti Rotor, Swinging Bucket) in a discontinuous density gradient (17–30%) of iodixanol (OptiPrep). Immediately after centrifugation each fraction was probed for mitochondria, peroxisomes, and Golgi and ER proteins as well as for the presence of the lysosomal membrane proteins LAMP-2, LAMP-1 and Hexosaminidase by Western blot (Supplementary Fig. 2A ).
5
Lysosomal Isolation and Proteomic Analysis
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Lysosomal extracts were prepared at day 6 post‐lentiviral transduction by density gradient ultracentrifugation using the Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo Scientific) according to the manufacturer's protocol. One readout constituted of six independent spheroid culture samples pooled together.
Presence of the lysosomes and purity of the collected fractions were verified using Western blot analysis with specific antibodies against lysosomes (LAMP2) and endoplasmic reticulum (Calnexin 1). Extracted lysosome pellets were stored at −80°C and further analysed by mass spectrometry. The experiment was performed two times.
Presence of the lysosomes and purity of the collected fractions were verified using Western blot analysis with specific antibodies against lysosomes (LAMP2) and endoplasmic reticulum (Calnexin 1). Extracted lysosome pellets were stored at −80°C and further analysed by mass spectrometry. The experiment was performed two times.
6
Lysosome Isolation and Purity Assessment
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Lysosome fraction was collected by using the Lysosome Enrichment Kit for Tissue and Cultured Cells according to the manufacturer's protocol (Thermo Scientific, 89839, Rockford, IL, USA). Cells (1 × 107 cells) were treated with IL-2(+) or IL-2(−) for 24 h, and cell pellets were used for lysosome isolation. Whole cell lysate before lysosome collection and isolated lysosome fractions were then subjected to western blotting analysis to check the purity of the isolated lysosome and measurement of SM by LC-MS/MS. Anti-Lamp2 (lysosome), anti-pan-cadherin (plasma membrane), and anti-GAPDH (cytosol) antibodies were used as markers of each organelle.
7
Isolation and Characterization of Lysosomes
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The isolation of lysosomes was preformed with Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo, Rockford, IL, USA). Two hundred micrograms of A549 cells was harvested and then homogenized. Homogenates were then centrifuged at 500 × g for 10 min at 4 °C and the supernatants were collected. The supernatants mixed with OptiPrep Cell Separation Media were overlayed on the top of a prepared discontinuous density gradient. Samples were centrifuged at 145 000 × g for 2 h at 4 °C to form several bands; the lysosomal and cytoplasmic bands were removed. The lysosomal band was mixed with two to three volumes of PBS, followed by centrifugation at 18 000 × g for 30 min at 4 °C. The lysosomes were lysed with 2% CHAPS in PBS. The cytoplasmic and lysosomal fractions were subjected to SDS-PAGE and immunoblotted for BAX and cathepsin B.
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