DC lysosomal extracts were obtained as previously described using the lysosome isolation kit (Sigma-Aldrich, St. Louis, MO)26 (link),36 (link). Briefly, DCs were lysed using 1X extraction buffer (Sigma-Aldrich) followed by homogenization with a PowerGen 700 homogenizer (Fisher Scientific, Pittsburgh, PA) using the 7 × 110 mm homogenizer tip passed through the cells 15–20 times to disrupt 75–80% of the cells. The homogenized cells were then centrifuged at 1000 × g for 10 min to remove intact cells and cellular debris. The first supernatant was removed and centrifuged at 20,000×g for 20 min to pellet lysosomes. Lysosome purity was verified by flow cytometry for lysosomal marker LAMP1 (BD Biosciences, San Jose, CA), which showed > 90% pure lysosomes from this procedure. The pellet containing lysosomes was resuspended and sonicated for 20 s at 40% power on a Model 300 VT Ultrasonic Homogenizer (BioLogics, Inc., Manassas, VA), resulting in lysosome extract (600 mg/ml).
Lysosome isolation kit
Manufactured by Merck Group
Sourced in United States
The Lysosome Isolation Kit is a laboratory tool designed to extract and purify lysosomes from cells. Lysosomes are organelles within cells responsible for the breakdown and recycling of cellular components. The kit provides the necessary reagents and protocols to isolate these subcellular structures for further research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Optiprep, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Powergen 700, by Thermo Fisher Scientific (1 mentions)
Lamp1, by BD (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Flag m2 bead, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail and phosphatase inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Optima max xp benchtop ultracentrifuge, by Beckman Coulter (1 mentions)
Ez link psoralen peg3 biotin, by Thermo Fisher Scientific (1 mentions)
Phospho irf3, by Cell Signaling Technology (1 mentions)
Rabbit polyclonal antibodies against irf3, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 555 conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
41 protocols using lysosome isolation kit
1
Isolation and Preparation of Lysosomal Extracts from Dendritic Cells
2
Cellular Fractionation and Lysosome Isolation
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Nuclear and cytoplasmic fractions were prepared using the NE-PER kit (Thermo Scientific, 78833) according to the manufacturer’s protocol. Lysosome isolation was performed using a lysosome isolation kit (Sigma-Aldrich, LYSISO1) according to the manufacturer’s manual.
3
Lysosome Isolation and Protein Analysis
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The ipsilateral cortexes from mice (n = 4 in each group) were lysed in RIPA buffer (Cat# P0013B, Beyotime) containing protease inhibitors and phosphatase inhibitor. A lysosome isolation kit (Cat# Lysiso1, Sigma-Aldrich, St. Louis, MO, USA) was used to obtain the lysosomes and the remaining cytoplasmic components in the brain tissue, and the cytoplasmic components were added to RIPA buffer for lysing to obtain cytoplasmic protein. The protein concentrations were determined using a BCA Protein Assay Kit (Cat# CW0014, CWBIO, Beijing, China). The protein sample lysates were electrophoretically separated on an SDS-PAGE (10% or 12%), then transferred from gels to 0.45 µm polyvinylidene fluoride membranes (Cat# IPVH00010, Millipore, Boston, MA, USA). The membranes with proteins were blocked for 1 h with 5% non-fat milk in TBST at room temperature and then incubated overnight at 4 °C with the following primary antibodies (Dilution: 1:1000). An enhanced chemiluminescence (Cat# WBKLS0500, Millipore, Boston, MA, USA) system was used to monitor the blot signals. All experiments were performed three times. Protein quantitative analysis was conducted using ImageJ software. All antibodies used in the method are shown in Table 1 .
4
Lysosome Enrichment and Isolation
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For each group, 2 × 108 cells were harvested and trypsinized. Lysosome isolation was performed according to the manufacturer’s description (Lysosome Isolation Kit; Sigma-Aldrich, LYSISO1). Briefly, cells were centrifuged at 600 × g for 5 min, resuspended in a 2.7 packed cell volume of 1 × extraction buffer, and homogenized in a glass Dounce homogenizer by 20 strokes. The nuclei were removed by centrifugation at 1,000 × g for 10 min. The post-nuclear supernatant was centrifuged at 20,000 × g for 20 min, and the resulting pellet, containing the crude lysosomal fraction, was resuspended in a minimal volume of 1 × extraction buffer (0.4 ml per 108 cells). A step gradient medium solution, including 27%, 22.5%, 19%, 16%, 12% and 8% was prepared according to the manufacturer’s recommendations, with 27% Optiprep Density Gradient Medium Solution at the bottom and 8% Optiprep Density Gradient Medium solution at the top of the tube. We isolated enriched lysosomes from the crude lysosomal fraction by density gradient centrifugation at 150,000 × g for 4 h on an OptiPrep (Sigma-Aldrich, LYSISO1) density gradient. Altogether, 0.5 ml fractions were collected starting from the top of the gradient. Each fraction was tested for protein concentration, ACP2 and NAGLU activity.
5
Macrophage-derived Extracellular Vesicles Isolation
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Macrophage FTV were isolated using procedures from previous reports with some modifications [19 (link)]. Briefly, nonpolarized macrophages, M1, M2 differentiated macrophages and HG treated macrophages were initially washed by serum-free cell culture medium, followed by treatment with trypsin. Subsequently, a low-speed centrifugation at 1000 × g for 10 min, then at 2000 x g for 20 min to remove cells and cellular debris. The collected cell culture medium was then mixed with an extraction buffer (Lysosome Isolation Kit, Sigma-Aldrich, USA), These crude FTV fraction were subjected to 150 000× g for 4 h ultracentrifugation at a multi-step 2%, 5%, 8%, 10%, 12%, 15%, 19%, 25%, 30% Optiprep density gradient medium solutions. The purified FTV fraction were then carefully collected and named M0-ftv, M1-ftv, M2-ftv, HG-ftv respectively. The prepared samples are subjected to further analysis or stored at -80 °C for subsequent processing. FTV size distribution and quantification of vesicles was analyzed by Nanoparticle tracking analysis (NTA) .
6
Immunoprecipitation and Mass Spectrometry Analysis of Lysosomal Proteins
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FLAG- Akt2 and HA-Phafin2 were transformed into 293T cells, treated with HBSS for 4 h (Sup. 1a-b ). The cells were harvested, rinsed twice with ice-cold phosphate-buffered saline. The lysosomal-enriched fraction was purified using the Lysosome Isolation Kit (LYSISO1, Sigma), precleaned with ProA/ProG beads, immunoprecipitated with FLAG-M2 beads (Sigma), and subjected to MS/MS analysis by LXQ (Thermo Fisher) equipped with nano-flow LC Magic (Michrom Bioresources, Inc.) [36 (link)]. The spectra for potential peptide that are enriched after autophagy induction were determined.
7
Isolation and Characterization of Cellular Organelles
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Cell lysates were prepared from primary cultured microglia, neurons, HEK293T cells or brain tissue by RIPA lysis buffer (Beyotime, China) with protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Nuclear and cytoplasmic extracts were prepared by the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, #78833) according to the manufacturer's instructions. Protein concentrations were determined using an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime, China) prior to Western blot or immunoprecipitation analysis. Lysosomes were isolated by a lysosome isolation kit (Sigma) with density gradient ultracentrifugation according to the manufacturer's description. Brain tissue was prepared from the ipsilateral parietal cortex (penumbra of the middle cerebral artery) or hippocampus and the corresponding area of the contralateral side 20 (link).
8
Isolation of Mouse Cerebella Lysosomes
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Fresh cerebella were dissected from mice that were starved overnight and killed the next morning. Then lysosome isolation by subcellular fractionation from the mice cerebella was performed with a lysosome isolation kit (Sigma-Aldrich) according to the manufacturer's manual. After a discontinuous iodixanol gradient centrifugation using Optima MAX-XP Benchtop Ultracentrifuge (Beckman Coulter) with MLS-50 rotor at 150,000 × g and 4°C for 4 h, the sample was divided into ten fractions (0.5 ml each) for further biochemical analyses.
9
Investigating Innate Immune Signaling Pathways
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Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R&D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3-Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers. Mouse antisera against human Mex3B were raised against recombinant human Mex3B fragments containing aa236-517 and aa1-517, respectively.
10
Synthesis and Characterization of Silica Nanoparticles
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n-Cetyltrimethylammonium
bromide (CTABr; purity ≥98%), tetraethyl orthosilicate (TEOS;
purity: 98%), sodium hydroxide (NaOH; purity ≥98%), polyethylenimine
(PEI; average molecular weight: 25 000 g/mol), hyaluronic acid
(HA; #53747), propidium iodide (PI; purity ≥94%), diamidino-2-phenylindole
dihydrochloride (DAPI; purity ≥98%), lysosome Isolation Kit
(LYSISO1), and tetramethylammonium hydroxide solution (TMAH: 25 wt
% in H2O) were purchased from Sigma-Aldrich (St. Louis,
MO). Tetramethylrhodamine-5-isothiocyanate (TRITC; T490), RNA labeled
with Cy3 (miRCy3, AM4621), DMEM/F12, RPMI 1640,l -glutamine,
penicillin-streptomycin (10000 U/mL), fetal bovine serum (FBS), formaldehyde
(28908), LysoTracker Deep Red (L12492), Hoechst33342, scramble miRNA
(4464058), and Lipofectamine 2000 were acquired from Thermo Fisher
Scientific (Waltham, MA). miR-200c-3p was acquired from Guangzhou
RiboBio CO (Guangzhou, China).
bromide (CTABr; purity ≥98%), tetraethyl orthosilicate (TEOS;
purity: 98%), sodium hydroxide (NaOH; purity ≥98%), polyethylenimine
(PEI; average molecular weight: 25 000 g/mol), hyaluronic acid
(HA; #53747), propidium iodide (PI; purity ≥94%), diamidino-2-phenylindole
dihydrochloride (DAPI; purity ≥98%), lysosome Isolation Kit
(LYSISO1), and tetramethylammonium hydroxide solution (TMAH: 25 wt
% in H2O) were purchased from Sigma-Aldrich (St. Louis,
MO). Tetramethylrhodamine-5-isothiocyanate (TRITC; T490), RNA labeled
with Cy3 (miRCy3, AM4621), DMEM/F12, RPMI 1640,
penicillin-streptomycin (10000 U/mL), fetal bovine serum (FBS), formaldehyde
(28908), LysoTracker Deep Red (L12492), Hoechst33342, scramble miRNA
(4464058), and Lipofectamine 2000 were acquired from Thermo Fisher
Scientific (Waltham, MA). miR-200c-3p was acquired from Guangzhou
RiboBio CO (Guangzhou, China).
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