Llome

Manufactured by Merck Group

Sourced in United States

LLOMe is a laboratory equipment product manufactured by Merck Group. It is a specialized tool designed for use in scientific research and analysis. The core function of LLOMe is to facilitate precise measurement and handling of small liquid volumes. Further details on the intended use or applications of this product are not available.

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Lab products found in correlation

Mli 2, by Bio-Techne (3 mentions) Nigericin, by Merck Group (2 mentions) Monensin, by Merck Group (2 mentions) Zymosan, by Merck Group (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Nocodazole, by Merck Group (2 mentions) Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Rapamycin, by Cayman Chemical (1 mentions) 96 well glass bottom plates, by PerkinElmer (1 mentions) Valinomycin, by Merck Group (1 mentions) Salinomycin, by Merck Group (1 mentions) Bafa1, by Selleck Chemicals (1 mentions) Pik 3, by Selleck Chemicals (1 mentions) Mk6 83, by Bio-Techne (1 mentions) Ml sa1, by Bio-Techne (1 mentions) Azd8055, by Selleck Chemicals (1 mentions) Anti fip200 17250 1 ap, by Proteintech (1 mentions) Anti α tubulin dm1a, by Merck Group (1 mentions) Anti lc3 l7543, by Merck Group (1 mentions)

Close spelling variants of this product

L-leucyl-L-leucine methyl ester (LLOMe) (6 citations) Leu‐Leu methyl ester hydrobromide (LLOMe, (4 citations) LLOMe (L7393) (2 citations)

16 protocols using llome

Visualizing Autophagy and Lysophagy

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Cells stably expressing tf-LC3 were cultured in 96-well glass-bottom plates (PerkinElmer), treated with growth medium with or without 250 nM Baf.A1 for 2 h, fixed with 4% PFA for 20 min, and then observed using CQ1 software (Yokogawa).
For lysophagy assays, cells stably expressing tf-Gal3 were cultured in 96-well glass-bottom plates, and treated with 1 mM LLOMe (Sigma-Aldrich, L7393) for 1 h. Then, LLOMe was washed out with DMEM and cells were incubated in DMEM without LLOMe for 0 or 10 h. After incubation for the indicated times, cells were fixed with 4% PFA for 20 min. CQ1 software (Yokogawa) was used to observe the cells and calculate the numbers of tf-LC3 and the mean intensity of tf-Gal3 dots per cell.

Oe Y., Kakuda K., Yoshimura S.I., Hara N., Hasegawa J., Terawaki S., Kimura Y., Ikenaka K., Suetsugu S., Mochizuki H., Yoshimori T, & Nakamura S. (2022). PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy. PLoS Genetics, 18(6), e1010264.

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Lysosomal Trafficking and Stress Assays

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All compound treatments were applied 3–4 weeks after reseeding of neurons in the final plate format. A 2 µM CZC-25146 (MedChemExpress, Monmouth Junction, NJ, USA) and 2 µM MLi-2 (MedChemExpress) treatment for lysosomal axonal trafficking experiments was applied for 48 h. For the LLOME (L-Leucyl-L-Leucine methyl ester)-mediated stress experiments, 1 µM LLOME (Sigma Aldrich) treatment was applied for 2 h. A 1 µM MLi-2 treatment was applied 90 min before LLOME treatment; hence, treatment was applied for 3.5 h in total. All treatments included the DMSO control that matched the maximum concentration and maximum treatment duration. Compounds were dissolved in maturation medium.

Bhatia P., Bickle M., Agrawal A.A., Truss B., Nikolaidi A., Brockmann K., Reinhardt L., Vogel S., Szegoe E.M., Pal A., Hermann A., Mikicic I., Yun M., Falkenburger B, & Sterneckert J. (2024). Axonal Lysosomal Assays for Characterizing the Effects of LRRK2 G2019S. Biology, 13(1), 58.

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FKBP-FRB Complex Regulation by Rapamycin

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For all of the experiments using the FKBP-FRB complex, rapamycin (Cayman Chemicals, cat no. 13346) was added at 200 nM for 15 min before fixation in 4% paraformaldehyde. MLi-2 (Tocris, cat. no. 5756) was used at 1 µM in dimethyl sulfoxide (DMSO) for 90 min, or LLOMe (Sigma-Aldrich, cat. no. L7393) was added at 1 mM in DMSO for 2 h before fixation or lysing cells for downstream analyses. Starvation experiments consisted of rinsing cells with phosphate-buffered saline, followed by incubation in Earle’s balanced salt solution for 3 h before fixation. Time points from 2 to 16 h of starvation were explored and all resulted in no significant lysosomal clustering in HEK293FT cells. The full experimental procedures and associated references can be found in SI Appendix, Materials and Methods.

Kluss J.H., Beilina A., Williamson C.D., Lewis P.A., Cookson M.R, & Bonet-Ponce L. (2022). Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes. Proceedings of the National Academy of Sciences of the United States of America, 119(43), e2205492119.

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Pharmacological Modulators of Cellular Processes

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BafA1, PIK-III, and AZD8055 were purchased from Selleckchem. ML-SA1 and MK6-83 were purchased from Tocris. Monensin, nigericin, salinomycin, valinomycin, and LLoMe were purchased from Sigma-Aldrich. C8 is available for purchase through ChemShuttle (catalog no. 187417).

Goodwin J.M., Walkup WG I.V., Hooper K., Li T., Kishi-Itakura C., Ng A., Lehmberg T., Jha A., Kommineni S., Fletcher K., Garcia-Fortanet J., Fan Y., Tang Q., Wei M., Agrawal A., Budhe S.R., Rouduri S.R., Baird D., Saunders J., Kiselar J., Chance M.R., Ballabio A., Appleton B.A., Brumell J.H., Florey O, & Murphy L.O. (2021). GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis. Science Advances, 7(40), eabj2485.

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Antibody Validation for LRRK2 and Lysosomal Proteins

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The following primary antibodies were used in this study: anti-LRRK2 (MJFF2 [c41-2]; Abcam), anti-human LAMP1 (D2D11; Cell Signaling Technology), anti-mouse LAMP1 (1D4B; Bio-Rad), anti-LC3 (PM036; MBL), anti-LC3 (L7543; Sigma-Aldrich), anti-GFP (A-11122; Invitrogen), anti-p62 (61832; BD Pharmingen), anti-galectin-3 (M3/38; BioLegend), anti-α-tubulin (DM1A; Sigma-Aldrich), anti-CatD (ERP3057Y; ab75852), anti-CatB (D1C7Y; Cell Signaling Technology), anti-Rab10 (D36C4; Cell Signaling Technology), anti-phospho-Thr73 Rab10 (MJF-R21 [ab230261]; Abcam), anti-ATG16L1 (D6D5; CST), anti-ATG5 (A0731; Sigma-Aldrich), anti-FIP200 (17250-1-AP; Proteintech), anti-ATG13 (SAB4200100; Sigma-Aldrich), and anti-Rab29 (MJF-R30-124; Abcam). The following reagents were used at final concentrations as indicated: CQ (50–100 μM; Sigma-Aldrich), LLOMe (1 mM; Sigma-Aldrich), and zymosan (Sigma-Aldrich).

Eguchi T., Sakurai M., Wang Y., Saito C., Yoshii G., Wileman T., Mizushima N., Kuwahara T, & Iwatsubo T. (2024). The V-ATPase–ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response. The Journal of Cell Biology, 223(3), e202302067.

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Drug Treatments for Cellular Processes

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Drug treatments were performed as follows unless otherwise specified. Leupeptin (Alfa Aesar, J61188) and pepstatin A (MP Biologicals, 195368) were used at 20 μM each for 24 h. Bafilomycin A1 (Alfa Aesar, J61835) was used at 500 nM for 4 h. LLOMe (Sigma, L7393) was used at 1 mM for 10 m unless otherwise indicated in the figure legend. Torin1 (Tocris, 4247) was used at 250 nM for 24 h, and rapamycin (Calbiochem, 553210) was used at 100 nM for 24 h. OSW-1 (a generous gift from Matthew Shair, Harvard University) was used at 10 nM for 8 h. CCCP (Sigma, C2759) was used at 10μM for 5 h. U18666A (Tocris, 1638) was used at 5 μg/ml for 24 h.

Davis O.B., Shin H.R., Lim C.Y., Wu E.Y., Kukurugya M., Maher C.F., Perera R.M., Ordonez M.P, & Zoncu R. (2020). NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C. Developmental cell, 56(3), 260-276.e7.

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Immunoblotting and Immunofluorescence Techniques

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For immunoblotting and immunofluorescence, the following antibodies were used in this study: anti-FLAG (mouse; Sigma-AldrichF1804; Sigma-Aldrich), anti-α-tubulin (mouse; B512; Sigma-Aldrich), anti-TBK1 (rabbit; EP611Y; Abcam), anti-p-TBK1 (Ser172; rabbit; D52C2; Cell Signaling Technology), anti-LAMP1 (mouse; sc-19992; Santa Cruz Biotechnology), anti-p62 (rabbit; PM045; MBL), anti-ubiquitin antibody, Lys48-specific (rabbit; 05-1307; Millipore), anti-ubiquitin antibody, Lys63-specific (rabbit; 05-1308; Millipore), and anti-polyubiquitin antibody FK2 (mouse; 0918-2; Nippon Bio-Test Laboratories). The secondary antibodies used for immunoblotting were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (111-035-003; Jackson ImmunoResearch) and HRP-conjugated goat anti-mouse IgG (115-035-003; Jackson ImmunoResearch). The secondary antibodies used for immunofluorescence were goat anti-rabbit Alexa Fluor 488 preabsorbed (ab1500085; Abcam) and goat anti-mouse IgG (H+L) cross-adsorbed Alexa Fluor 568 (A11004; Invitrogen). LLOMe was purchased from Sigma-Aldrich. Depending on the experiment, cells were treated for 1 h with 10 µM BAPTA-AM (Dojindo) or for 20 h with 250 nM bafilomycin A1 (Cayman Chemical).

Shima T., Ogura M., Matsuda R., Nakamura S., Jin N., Yoshimori T, & Kuma A. (2023). The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps. The Journal of Cell Biology, 222(12), e202204048.

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HeLa Cell Culture and Transfection Protocol

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Human HeLa “Kyoto” cells were maintained in DMEM (Gibco) medium, supplemented with 10% fetal bovine medium, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37°C supplemented with 5% CO₂. A stable HeLa cell line expressing CHMP4B-eGFP was obtained from Anthony A. Hyman (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany) and was maintained with the media described before plus G418 (geneticin) 300 µg/ml. These cells were routinely tested mycoplasma-negative by GATC Biotech.
The lysomotropic drug LLOMe was purchased from Sigma-Aldrich (L7393). LLOMe was dissolved in DMSO and properly aliquoted and stored at −20°C.
For transient expression experiments, Lipofectamine 3000 from Thermo Fisher Scientific was used for live-imaging and immunofluorescence experiments, and TransIT-LT1 Transfection Reagent (MIR2300) from Mirus was used for quantification experiments.

Pfitzner A.K., Zivkovic H., Bernat-Silvestre C., West M., Peltier T., Humbert F., Odorizzi G, & Roux A. (2023). Vps60 initiates alternative ESCRT-III filaments. The Journal of Cell Biology, 222(11), e202206028.

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Investigating Lysosomal Calcium Regulation

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Reagents and chemicals used were LLOMe (L7393; Sigma-Aldrich), GPN (SC-252858; Scbt), BafA1 (1334; Tocris), PP242 (4257; Tocris), Monensin (M5273; Sigma-Aldrich), human serum (H2918; Sigma-Aldrich), Zymosan (Z4250; Sigma-Aldrich), murine IFNγ (315-05; Peprotech), DAPI (D9542; Sigma-Aldrich), IN-1 (17392; Caymen Chemical), MLi2 (5756; Tocris), and saliphenylhalamide (SaliP), and TRPML1 agonist C8 and ATG7 inhibitor ATG7-IN-3 were kindly provided by Casma Therapeutics. BAPTA-AM (B1205; Thermo Fisher Scientific), nocodazole (M1404; Sigma-Aldrich), GFP-Trap (gtma-20), and control magnetic agarose beads (bmab-20) were obtained from Chromotek.

Cross J., Durgan J., McEwan D.G., Tayler M., Ryan K.M, & Florey O. (2023). Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy. The Journal of Cell Biology, 222(12), e202303078.

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Lysosomal Damage and Autophagic Flux

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For lysosomal damage experiments, BMMs were incubated with LLOMe (Sigma) at the indicated concentrations for 2 hours prior to fixation. For the autophagic flux experiments, Mtb-infected BMMs were treated with vehicle control (DMSO) or bafilomycin A1 (Sigma) at the indicated concentrations for 3 hours prior to fixation.

Morrison H.M., Craft J., Rivera-Lugo R., Johnson J.R., Golovkine G.R., Bell S.L., Dodd C.E., Van Dis E., Beatty W.L., Margolis S.R., Repasy T., Shaker I., Lee A.Y., Vance R.E., Stanley S.A., Watson R.O., Krogan N.J., Portnoy D.A., Penn B.H, & Cox J.S. (2023). Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens. PLOS Pathogens, 19(6), e1011088.

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