Cells were harvested 72 h after transfection with siRNAs and lysed in RIPA buffer composed of 25 mM Tris‐HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P‐40, 1% Na‐deoxycholate, and 0.1% SDS. For detection of LC3A/B, cells were lysed in lysis buffer consisting of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.2 mM EDTA, 300 mM NaCl, and 1% Nonidet P‐40. Both lysis buffers were supplemented with protease inhibitors (complete EDTA‐free protease inhibitor cocktail, Roche) and phosphatase inhibitors (PhosStop, Roche). For detection of endogenous UBE2QL1, cells were harvested in lysis buffer of the lysosome enrichment kit (ThermoFisher Scientific #89839) and homogenized with a dounce tissue grinder. Samples were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Amersham, GE Healthcare). Immunoblot analysis was performed with the indicated antibodies and visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce).
Lysosome enrichment kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lysosome Enrichment Kit is a laboratory tool designed to isolate and enrich lysosomal fractions from cells or tissues. Lysosomes are specialized organelles within eukaryotic cells that play a crucial role in the degradation and recycling of cellular components. The kit provides a reliable and efficient method for the isolation and purification of lysosomes, enabling researchers to study their structure, function, and composition.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay, by Bio-Rad (2 mentions)
Optiprep, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
Qproteome mitochondrial isolation kit, by Qiagen (1 mentions)
Sw 60 ti swinging bucket rotor, by Beckman Coulter (1 mentions)
Optiprep gradient media, by Merck Group (1 mentions)
Topfluor cholesterol, by Merck Group (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Acid phosphatase assay kit, by Merck Group (1 mentions)
Cell fractionation kit, by Abcam (1 mentions)
Human tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides lps from escherichia coli 0111 b4, by Merck Group (1 mentions)
Ecl plus western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Cell Signaling Technology (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Simvastatin, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
7β hydroxycholesterol, by Merck Group (1 mentions)
29 protocols using lysosome enrichment kit
1
Western Blot Analysis of Cell Lysates
2
Isolation of Lytic Granules from NK Cells
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Lytic granules were isolated from YTS NK cell lines for lipidomic analyses using the Thermo Fisher Scientific lysosome enrichment kit as previously described [57 (link)]. Briefly, 108 YTS cells were harvested from culture and then mechanically lysed using a Dounce homogenizer. The homogenized lysates were centrifuged at 500 × g for 10 min to remove the nuclei. Supernatant was collected and carefully overlaid on an 8% to 27% Optiprep gradient and centrifuged at 145,000g for 2 h at 4°C in a Beckman Optima centrifuge using a SW 50.1 rotor. After centrifugation, the lysosome band was carefully removed from the Optiprep gradients and washed twice with PBS at 18,000 × g for 30 min at 4°C to remove the Optiprep in a Beckman Optima centrifuge using a SW 50.1 rotor. Purified lytic granules were kept in −80°C for future use.
3
Subcellular Fractionation of Spinal Cord
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Around 5-mm fragments of spinal cord tissue centered on the injury site or corresponding site in sham animals were collected from sham mice, and at 2 or 24 h after and homogenized in ice-cold buffered solution containing 0.32 M sucrose, 10 mM Hepes with the addition of protease and phosphatase inhibitors24 (link). Homogenates were centrifuged at 800 × g for 10 min at 4 °C to pellet down the nuclei. Supernatants were sequentially centrifuged at 20,000 × g for 20 min at 4 °C to pellet the heavy membrane/crude lysosomal fractions and at 100,000 × g for 1 h at 4 °C to pellet light membrane fractions. Both supernatant and suspended pellet fractions were recentrifuged to minimize cross contamination from the different subcellular fractions. All pellets were resuspended in homogenization buffer. Protein concentration was estimated using BCA reagent; samples were analyzed by Western Blot.
For isolation of purified lysosome samples, after the 5 mm segment of the spinal cord was extracted, the lysosome enrichment kit (Thermo Scientific, Cat.) was used according to the manufacturer’s instructions to obtain a purified form of lysosome for downstream testing with Western Blot analysis and MASPEC lipidomic.
For isolation of purified lysosome samples, after the 5 mm segment of the spinal cord was extracted, the lysosome enrichment kit (Thermo Scientific, Cat.) was used according to the manufacturer’s instructions to obtain a purified form of lysosome for downstream testing with Western Blot analysis and MASPEC lipidomic.
4
Lysosomal Isolation from Hepa-1c1c7 Cells
5
Subcellular Fractionation of SH-SY5Y Cells
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Isolation of mitochondrial and lysosomal fractions from SH-SY5Y WT and SACS KO cell lysates was performed using the Qproteome Mitochondrial Isolation Kit (Qiagen, Hilden, Germany) and the Lysosome Enrichment Kit (Thermo Scientific™, Waltham, MA, USA), respectively, according to the manufacturers’ instructions. About 50 mg of cells obtained from four confluent F75 flasks (harvested without trypsin) were processed for organelle enrichment. The pellets containing mitochondria or lysosomes were solubilized in a lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, and protease inhibitors. The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
6
Lysosomal Cathepsin B Release Assay
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Lysosomes were isolated using a Lysosome Enrichment Kit (Thermo Fisher) according to the manufacturer’s protocol. The equal portions of isolated lysosomes were incubated as indicated for 12 h at 37 °C and then was centrifuged at 15,000 × g for 30 min at 4 °C to pellet intact lysosomes. The release of cathepsin B into the supernatant was determined (Ex = 380 mm, Em = 460 mm) after a 2 h incubation with 200 μM fluorogenic Cathepsin B Substrate III (Z-Arg-Arg-AMC).
7
Subcellular Fractionation for Lysosome Enrichment
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To perform lysosome enrichment, subcellular fractionation was carried out using the Lysosome Enrichment Kit (Thermo Scientific). Briefly, the cell pellet was resuspended in PBS and homogenized with a dounce homogenizer on ice (~20 strokes). To confirm cell lysis, microscopic examination of homogenate was done by adding 0.5% trypan blue dye. The homogenate was subjected to centrifugation at 500×g for 10 min at 4 °C, and post-nuclear supernatant (PNS) was diluted in OptiPrep gradient media (Sigma-Aldrich) to a final concentration of 15% OptiPrep. The sample was then carefully overlayered on the top of a discontinuous density gradient (17, 20, 23, 27, and 30%). The gradient was subjected to ultracentrifugation at 145,000×g in an SW60 Ti swinging bucket rotor (Beckman Coulter) for 4 h at 4 °C. After the spin, eight fractions of 400 µl each were collected from top to bottom. The fractions were spun again at 18,000×g for 20 min in an SW41 Ti rotor at 4 °C, and the resulting pellet was suspended in 4X SDS-sample buffer, boiled for 10 min, and analyzed by SDS-PAGE and immunoblotting.
8
Subcellular Fractionation of Lysosomes
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Using the Lysosome Enrichment Kit (Thermo Scientific), subcellular fractionation was performed. Briefly, the cell pellet was rinsed in PBS and homogenized with 20 strokes of Dounce homogenizer on ice. The homogenate was centrifuged at 500 × g for 10 min at 4 °C, and the post-nuclear supernatant was diluted with OptiPrep gradient medium (Sigma-Aldrich) to a final concentration of 15% OptiPrep. The sample was carefully layered on a discontinuous density gradient (17%, 20%, 23%, 27%, and 30%). The gradient was ultracentrifuged at 1,45,000 × g for 4 h at 4 °C in a SW60 Ti swinging bucket rotor (Beckman Colter). After centrifugation, nine 400 µL fractions were collected from the top down. The collected fractions were centrifuged at 18,000 × g for 20 min in an SW41 Ti rotor at 4 °C, and the obtained pellet was suspended in Laemmli buffer, boiled for 10 min, and analyzed using SDS-PAGE and immunoblotting.
9
Lysosome-Mitochondria Interaction Assay
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Cells were treated with TopFluor cholesterol (5 μmol/L, 810255P, Sigma‐Aldrich) before lysosome isolation by density gradient centrifugation using a Lysosome Enrichment kit (89839, Thermo Fisher Scientific). The lysosome fractions were diluted with a reconstitution buffer (250 mmol/L sucrose, 1 mmol/L DTT, 1 mmol/L MgCl2, 50 mmol/L KCl, and 20 mmol/L HEPES, pH 7.2). For mitochondria isolation, cells stably expressing 3× HA‐EGFP‐OMP25 (#83356, Addgene) were homogenized in ice‐cold PBS with homogenizer and spun down at 800 × g at 4°C for 10 min. The supernatant was spun down at 8,000 × g at 4°C for 10 min to collect the pellets. The pellets were then suspended with reconstitution buffer. Isolated mitochondria and lysosomes were incubated in 1 mg/mL cytosol of Huh7 cells, 1 mmol/L ATP, 1 mmol/L GTP, and an ATP‐regenerating system (30 mmol/L creatine phosphate, 0.05 mg/mL creatine kinase) at 37°C for 30 min. To isolate mitochondria from the reaction, pre‐washed anti‐HA magnetic beads were added and incubated for 5 min with rolling (60 rpm). The beads were then washed with PBS 3 times and eluted with 80% methanol extraction buffer. The eluates were subjected to microscopy or a microplate reader (SpectraMAX i3×, Molecular Devices, San Jose, CA, USA).
10
Isolation and Characterization of Lysosomes
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For the isolation of lysosomes, approx. 50 million cells were collected after 2 h of exposure to CuO NPs at the indicated concentrations. Cells were then processed using the lysosome enrichment kit (Thermo Fisher Scientific). Briefly, cell extracts were prepared in lysosome enrichment reagent A by homogenizing the cell pellet using a pre-chilled Dounce tissue grinder on ice. Lysed cells were then transferred into a microcentrifuge tube and lysosome enrichment reagent B was added. Next, supernatants were collected in fresh tubes by centrifugation (500×g for 10 min at 4 °C) and proceeded with the density gradient ultracentrifugation at 145,000×g for 2 h at 4 °C. The upper lysosome fraction was collected and mixed with PBS. Next, the diluted lysosome fraction was centrifuged at 18,000×g for 30 min at 4 °C and the pelleted lysosomes were stored at − 20 °C until further use. Lysosomal integrity in isolated samples was determined by measuring acid phosphatase activity using the acid phosphatase assay kit (Sigma) and the results confirmed that the isolated lysosomes remained intact (data not shown).
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