Dextran release assay was performed in H4 cells expressing RFP-LAMP1 as described previously [45 ]. 5 × 104 cells were seeded in glass bottom plates (Mattek Corp., P35G-1.0–14-C). After 24 h media was replaced with media containing 40 μg/ml of 3 kDa Alexa fluor 488-dextran (Thermo Fisher Scientific, D34682); after 16–18 h cells were washed twice with PBS and incubated with fresh phenol red free media for 2 h to achieve maximum uptake of dextran into the lysosomes. Cells were then treated with C1P or amyloid β in phenol red free media and visualized using fluorescent Nikon Ti-E inverted microscope at 60x at the end of the incubation. Cells were also treated with 2 mM L-leucyl-L-leucine-methyl ester (LLOME) (Sigma-Aldrich, L7393) for 2 h as a positive control. Images were processed and quantified using Nikon Elements software as above. Pearson’s correlation was used to quantify co-localization between lysosomes (RFP) and dextran (Alexa Fluor 488 in the FITC channel). To further assess lysosomal size and dextran loading, intensity profiles across selected cells were analyzed: lysosomes were identified as areas with RFP intensity > 1.5 average; length of continuous RFP > 1.5 profile segments was used to estimate the size of lysosomes. Dextran loaded lysosomes were defined as lysosomes with GFP intensity > 2.0 average.
L leucyl l leucine methyl ester llome
Manufactured by Merck Group
Sourced in United States
L-leucyl-L-leucine methyl ester (LLOMe) is a chemical compound used in laboratory research. It is a dipeptide, consisting of two amino acids, leucine, linked by a peptide bond. LLOMe is utilized as a tool compound in various experimental studies, but its core function is not to be interpreted or extrapolated beyond its purpose as a research tool.
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Lab products found in correlation
D1817, by Thermo Fisher Scientific (2 mentions)
Peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions)
Fm4 64, by Thermo Fisher Scientific (2 mentions)
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (2 mentions)
Lysotracker, by Thermo Fisher Scientific (2 mentions)
Tritc dextran, by Thermo Fisher Scientific (2 mentions)
Ni nta agarose, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 tfp ester, by Thermo Fisher Scientific (2 mentions)
Alexa555 egf, by Thermo Fisher Scientific (2 mentions)
L7528, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Dextrin sepharose high performance, by GE Healthcare (2 mentions)
Alexa 647 egf, by Thermo Fisher Scientific (2 mentions)
E35351, by Thermo Fisher Scientific (2 mentions)
E35350, by Thermo Fisher Scientific (2 mentions)
Mbptrap hd 5ml columns, by GE Healthcare (2 mentions)
Dspe peg biotin, by Avanti Polar Lipids (2 mentions)
Lissamine rhodamine b sulfonyl 18 1 rhod pe, by Avanti Polar Lipids (2 mentions)
Alexa fluor 488 dextran, by Thermo Fisher Scientific (1 mentions)
6 protocols using l leucyl l leucine methyl ester llome
1
Dextran Release Assay in Lysosomal Trafficking
2
Fluorescent Probes for Cell Imaging
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We obtained FM4-64, LysoTracker, Alexa 647-EGF, Alexa-555 EGF and TRITC-dextran (T13320, L7528, E35351, E35350 and D1817, respectively) from Thermo Fisher Scientific (Waltham, MA), as well as EZ-Link Sulfo-NHS-SS-Biotin, Alexa Fluor 488 TFP ester, Ni-NTA Agarose, 7 kDa MWCO Zeba Spin Desalting Columns and Hoechst 33342. Peroxidase-conjugated secondary antibodies were from Bio-Rad Laboratories (Hercules, CA), MBPTrap HD 5mL columns, Glutathione Sepharose 4B and Dextrin Sepharose High Performance from GE Healthcare (Anaheim, CA), and the cOmplete Protease Inhibitor Cocktail from Roche (Basel, Switzerland). We obtained DOPC, DSPE-PEG-Biotin, DOPE, DOPS and lissamine rhodamine B sulfonyl (18:1) Rhod PE from Avanti Polar Lipids (Alabaster, AL, and the lysosomotropic reagent L-leucyl-L-leucine methyl ester (LLOMe) from Sigma-Aldrich (St. Louis, MO). Other reagents and chemicals were obtained from Sigma-Aldrich (St. Louis, MO)
3
Fluorescent Probes for Cell Imaging
4
Inhibition of Autophagy Pathways
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Cells were grown to 80% confluence for 24 h in 6-/24-/96-well plates. Twenty-four h after seeding, the culture media was replaced with media containing the indicated treatments and the cells were incubated for different times according to the experiment: bafilomycin A1 100 nM (LC Laboratories, Woburn, MA, USA, B1080), rapamycin 1 µM (LC Laboratories, Woburn, MA, USA, R-5000), L-leucyl-L-leucine methyl ester (LLOMe) 1 mM (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany, L7393) and chloroquine (CQ) 50 μM (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany, C6628).
5
Galectin-3 Localization in Oxidative Stress
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RAW cells were seeded in DMEM supplemented with 5% FBS for 24 h at 37°C in 5% CO2. Cells were transfected with Galectin-3-GFP plasmid (0.5 μg) using FuGene HD (Promega, Madison, WI) with a ratio of 3:1 for 24 h at 37°C in 5% CO2. Post-transfection, cells were treated with 1 mM H2O2 for 40 min, 1 μM rotenone for 60 min or for 2 h with 0.5 mM L-leucyl-L-leucine methyl ester (LLOMe; L7393, Sigma-Aldrich).
6
Cellular PtdIns(3,5)P2 Depletion and ROS Generation
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Apilimod (Toronto Research Chemicals, Toronto, ON) was used at 20 nM for 40 min, unless otherwise indicated, to deplete cellular PtdIns(3,5)P2. H2O2 (Bio Basic, Markham, ON) was used as indicated. Rotenone, 1-chloro-2,4,-dinitrobenzene (CDNB), auranofin, and monocholorobimane (MCB; all from Sigma-Aldrich, Oakville, ON) were used as indicated to generate ROS by respectively inhibiting mitochondrial respiratory chain complex, thiol group, thioredoxin reductase, or glutathione. Bovine liver catalase (Sigma-Aldrich) and N-acetyl-L-cysteine (NAC) (Bio Basic) were used as anti-oxidants. Paclitaxel and nocadozole (both from Sigma-Aldrich) were used at 1 or 10 μM and 5 or 10 μM to stabilize and depolymerize microtubules, respectively. Latrunculin A (Abcam, Toronto, ON) and cytochalasin D (EMD Millipore, Toronto, ON) were used at 1 μM and 5 μM, respectively to depolymerize actin. Ikarugamycin (Sigma-Aldrich) and dyngo-4A (Abcam, Cambridge, MA) used to inhibit clathrin and dynamin respectively. BAPTA-AM (Sigma-Aldrich) was used to chelate intracellular calcium and Fluo4-AM (ThermoFisher, Burlington, ON) was used as a fluorescent Ca2+ probe. As a positive control for lysosome damage, we treated cells for 2 h with 0.5 mM L-leucyl-L-leucine methyl ester (LLOMe; L7393, Sigma-Aldrich).
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