To induce lysosomal damage, HeLa cells were treated with 250 μM Leu‐Leu methyl ester hydrobromide (LLOMe, Sigma) for indicated times or 8 μM Terfenadine (Sigma) for 24 h. Treatment with Alexa‐488 labeled Tau fibrils was done as described before 15 . For depolarization of mitochondria, cells were treated with 10 μM Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP, Sigma‐Aldrich) for 4 h. Cells were treated with 200 nM Bafilomycin A1 (Biomol) for 5 h or 1 μM Torin1 (Tocris Bioscience) for 2 h. Cell viability was measured using the 96® AQueous One Solution Cell Proliferation Assay (Promega).
Leu leu methyl ester hydrobromide llome
Manufactured by Merck Group
Leu‐Leu methyl ester hydrobromide (LLOMe) is a chemical compound used in laboratory research. It functions as a lysosomal protease inhibitor, which can be used to study cellular processes involving lysosomes. The compound has a specific chemical structure and is commonly utilized in experimental settings, though its precise applications may vary depending on the research objectives.
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Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Torin1, by Bio-Techne (1 mentions)
Terfenadine, by Merck Group (1 mentions)
Carbonyl cyanide 3 chlorophenylhydrazone cccp, by Merck Group (1 mentions)
96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Acridine orange, by Thermo Fisher Scientific (1 mentions)
Ca 074 me, by Merck Group (1 mentions)
Ethidium homodimer 1, by Thermo Fisher Scientific (1 mentions)
Ultrapure lps, by InvivoGen (1 mentions)
Z vad fmk, by InvivoGen (1 mentions)
Scrambled sirna, by Integrated DNA Technologies (1 mentions)
Il 1β elisa kit, by Thermo Fisher Scientific (1 mentions)
Recombinant il 1β, by Thermo Fisher Scientific (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
D9542, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Sorafenib tosylate, by MedChemExpress (1 mentions)
4 protocols using leu leu methyl ester hydrobromide llome
1
Inducing Lysosomal and Mitochondrial Damage
2
Investigating Inflammatory Pathways
3
Acridine Orange Assay for Lysosomal Destabilization
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For evaluation of lysosomal destabilization, cells were incubated for 15 min with acridine orange (1 μg/ml, 113000, Sigma‐Aldrich), washed three times, and then stimulated with either lecithinase or 1 μM Leu‐Leu methyl ester hydrobromide (LLOMe, L7393, Sigma‐Aldrich). For inhibition experiments, BMDMs loaded with acridine orange were pretreated for 30 min with inhibitors described earlier. Lysosomal destabilization was assessed by flow cytometry as loss of emission at 600–650 nm. For quantification of the kinetics of lysosomal destabilization and cell death, cells were loaded with acridine orange as described above and then treated with either lecithinase or LLOMe or transfected with LPS over different time points. The cells were washed and resuspended in FACS buffer in the presence or absence of DAPI (0.5 μg/ml, D9542, Sigma‐Aldrich). An LSRII (BD Biosciences) was used for flow cytometry. Data were acquired by DIVA (BD Biosciences) and analyzed with the FlowJo v10.7 software (Tree Star).
4
Mechanistic Insights into Autophagy Modulation
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Transfections were done using Lipofectamine 3000 (Thermo Fisher, L3000008) for plasmid DNA. Doxorubicin (Sigma-Aldrich, D1515) was diluted in water to 5 mM and used at 1 μM for 48 h. Bafilomycin A1 (BafA; Sigma-Aldrich, B1793) was diluted in DMSO to 200 μM and used at 100 nM for 4 h. Chloroquine (CQ; Sigma-Aldrich, C6628) was diluted in DMSO to 50 mM and used at 20 μM for 4 h. Rapamycin (Sigma-Aldrich, R0395) was diluted in ethanol to 2 mM and used at 200 nM for 4 h. Leu-Leu methyl ester hydrobromide (LLOMe; Sigma-Aldrich, L7393) was diluted in DMSO to 1 M and used from 0.001 to 1.4 mM for 6 h. Sorafenib tosylate (MedChemExpress, HY-10201A) was used from 5 to 14 μM for 24 h in vitro and 100 mg/kg in vivo daily. FITC-dextran (Sigma-Aldrich, FD40S) was directly diluted in the medium to 0.1 mg/mL. MTT (Thermo Fisher, M6494) was diluted in PBS (137 mM NaCl, 2.7 mM KCl, 12.5 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) to 5 mg/mL. For high starvation, cells were washed twice with PBS and starved in low glucose DMEM lacking amino acids (USBiological, D9800-13) and serum. To overexpress the TFEB variant with pronounced nuclear localization characteristics [45 (link)] the plasmid pCIP-caTfeb (Addgene, 79,013; deposited by Reuben Shaw) was transfected into Huh-7 VTRNA1-1 KO cells.
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