Magic Red Cathepsin B Assay was purchased from Immunochemistry Technologies (Bloomington, MN, USA). Lyso-Tracker Red and chloroquine (CQ) were from Sigma-Aldrich (St. Louis, MO, USA). U0126 and NAC (N-acetyl-cysteine) were obtained from Med Chem Express (Monmouth Junction, NJ, USA). GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). All remaining cell culture reagents were purchased from Life Technologies (Rockville, MD, USA). PVDF membranes were from Millipore (Burlington, MA, USA).
Lysotracker red
Manufactured by Merck Group
Sourced in United States
LysoTracker Red is a fluorescent dye that selectively labels acidic organelles, such as lysosomes, in live cells. It is a useful tool for the visualization and study of lysosomal structures and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
N acetylcysteine nac, by MedChemExpress (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Chloroquine, by Merck Group (1 mentions)
U0126, by MedChemExpress (1 mentions)
Magic red cathepsin b assay, by ImmunoChemistry Technologies (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Mitotracker green, by Merck Group (1 mentions)
Mitotracker red, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Blasticidin s hcl, by Thermo Fisher Scientific (1 mentions)
Thermolysin from bacillus thermoproteolyticus, by Merck Group (1 mentions)
Polyethyleneimine, by Polysciences (1 mentions)
Puromycin, by InvivoGen (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
9 protocols using lysotracker red
1
Cathepsin B Lysosomal Assay
2
Mitochondria and Lysosome Staining
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The mitochondria and lysosomes were visualized by MitoTracker™ Green FM (M7514, Invitrogen, Thermo Fisher Scientific, Inc.), MitoTracker™ Red CMXRos (M7512, Invitrogen, Thermo Fisher Scientific, Inc.) and LysoTracker™ Red DND-99 (L7528, Invitrogen, Thermo Fisher Scientific, Inc.) and followed the manufacturer’s instructions. Briefly, the targeted cells were exposed to MitoTracker Green or MitoTracker Red, LysoTracker Red, and Hoechst 33,342 (B2261, Sigma‒Aldrich, Merck) for 30 min, washed and observed by microscopy or confocal microscopy as described in a previous study [19 (link)].
3
SARS-CoV-2 Spike RBD Binding Assay
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Tubeimosides I (Cas No. 102040-03-9), Tubeimosides II (Cas No. 115810-12-3), and Tubeimosides III (Cas No. 115810-13-4) were purchased from Chengdu Biopurity Phytochemicals Ltd, China; U18666A (Cat No. 50205-0551) was from Cell Signaling Technology; Cathepsin B substrate (Cat No. sc-215529) and Cathepsin L substrate (Cat No. sc-3136) were from Santa Cruz Biotechnology; Filipin III (Cat No. GC12048) and CA074 (Cat No. GC15917) were from GLPBIO; EIPA (Cat No. HY-101840) and EST (E-64-d) (Cat No. HY-100229), and cholesterol (Cat No. HY-N0322) were from MedChemExpress; N-Dodecyl-β-D-maltoside (DDM) (Cat No. D4641), recombinant soluble ACE2 (Cat No. SAE0064), thermolysin from Bacillus thermoproteolyticus (Cat No. P1512), and anti-protease inhibitor cocktail (Cat No. P8340) were from Sigma Aldrich; Lyso-Tracker Red (Cat No. C1046), phosphoramidon (Metalloproteinase inhibitor) (Cat No. SG2024), and acetate buffer pH5.2 (Cat No. ST351) were from Beyotime Biotechnology; Lipofectamine 3000 (Cat No. L3000015) and Blasticidin S HCl (R21001) were from ThermoFisher; Puromycin (Cat No. ant-pr-1) was from InvivoGen; polyethyleneimine (Cat No. 23966-1) was from Polysciences, Inc; recombinant NPC1-C protein (Cat No. 16499-H32H) and recombinant SARS-CoV-2 spike RBD protein (Cat. No. 40592-V08H) were from SinoBiological.
4
Stem Cell Differentiation Pathway
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Lyso-tracker red, ascorbic acid (vitamin C, Vc), propidium iodide (PI), sodium selenite and DAPI were purchased from Sigma (MO, USA). All antibodies were purchased from CST (MA, USA). Fetal bovine serum (FBS) was purchased from Gibco. siRNA was provided by RIBRIO Co., Ltd (Guangzhou, China) directly against the sequence of siSox2: 5′-CCCGCAUGUACAACAUGAUUU-3’.
5
Visualizing Acidic Compartments with LysoTracker
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In order to visualize acidic compartments, cells were stained with LysoTracker Red DND-99 (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cells grown on 12 mm glass coverslips (Matsunami Glass Industry, Osaka, Japan, Cat# 1-S) coated with 0.2% gelatin from porcine skin (Sigma Aldrich, Cat# G2500) were incubated with 75 nM LysoTracker Red for 30 min at 37 °C in the dark. Next, the cells were rinsed once with PBS-C/M and fixed with 4% paraformaldehyde in PBS-C/M for 10 min at room temperature. The fixed samples were then quenched with 50 mM NH4Cl in PBS-C/M for 10 min, followed by blocking with PBS-C/M containing 0.5% BSA, 0.5% saponin, and 0.2 mg/ml sodium azide for 30 min. The samples were then subjected to immunostaining as described above. The images were acquired with a 40 × objective lens on a Zeiss LSM700 microscope (Carl Zeiss, Germany).
6
Apoptosis Induction Analysis Protocol
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Lysotracker red, sodium selenite (Na2SeO3), Hoechst 33342, propidium (PI), and ascorbic acid (Vc) were obtained from Sigma (MO, USA). Annexin V/PI was purchased from Beyotime. Fetal bovine serum (FBS) was obtained from Gibco. All antibodies were purchased from CST (MA, USA). The sequence of MEF2D-siRNA was as follows: GCAACAGCCTAAACAAGGT.
7
Visualization of Autophagic Vesicles and Lipid Droplets
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Autophagic vesicles and intracellular LD were detected based on the protocols of Klionsky et al. [5 (link)] as described in our previous study [24 (link)]. For detection of autophagic vesicles, cells were incubated with 1 mM acridine orange (AO, Sigma, USA), 50 mM monodansylcadaverine (MDC, Sigma, USA), or 50 nM LysoTracker Red (Sigma, USA) for 30 min. Then, they were washed three times in the ice-cold PBS. For intracellular LD staining, cells were incubated with 5 mg/ml Bodipy (Invitrogen, USA) for 30 min, and then washed three times in the ice-cold PBS. Fluorescence was imaged using laser scanning confocal microscopy (Leica, German) and flow cytometry (Beckman, USA) was used to determine fluorescence intensities.
8
Visualizing Garland Cell Endocytosis
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For LysoTracker Red staining, late L3 stage larval garland nephrocytes were dissected in cold Shields and Sang M3 medium (Sigma Aldrich), and then incubated in medium containing LysoTracker Red (1:1000, Thermo Fisher) for 5 min at room temperature (RT). Samples were rinsed 3 times and photographed immediately. For dextran uptake assay, late L3 stage larval garland cells were dissected in ice cold M3 medium, and then incubated in medium supplemented with Alexa Fluor 568 conjugated dextran (1 mg/ml, fixable, 10000 Da, Molecular Probes) for 5 min at RT, rinsed 3 times and fixed with 4% formaldehyde (FA) in PBS (30 min at RT). Nuclei were counterstained with DAPI in both cases. Pictures were taken on a microscope (AxioImager.Z1; Carl Zeiss) equipped with a grid confocal unit (ApoTome1; Carl Zeiss), using 40×, 0.75 NA (air), and 63×, 1.4 NA (oil) objectives in Lysotracker and dextran experiments, respectively, a CCD camera (AxioCam MRm; Carl Zeiss), and AxioVision software (Carl Zeiss).
9
Lysotracker Staining of Embryos
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Embryos were collected in PBS and immediately incubated in lysotracker red (Sigma) in PBS with calcium and magnesium for 30 mins at 37 o C. Embryos were then washed in PBS and fixed in 4% paraformaldehyde overnight at 4 o C. Embryos were washed in PBS, transferred to absolute methanol in graded steps and cleared in benzyl alcohol/benzyl benzoate (BABB) solution to visualize staining.
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