For the staining of vacuole, the 3- to 5-day-old PDA cultures incubated at 28 °C were stained with 5 μM of 7-amino-4-chloromethylcoumarin (CMAC) (Thermo Fisher, Waltham, MA, USA) for 60 min on ice. After three washes with phosphate buffered saline, the stained hyphae were observed under a fluorescence microscopy [48 (link)]. For the staining of lipid droplets (LDs) and autophagosomes, hyphal samples were stained with 10 µg/mL of boron dipyrromethene (BODIPY) dye (Sigma-Aldrich, USA) and 10 µg/mL of monodansylcadaverine (MDC, Sigma-Aldrich, USA), respectively, for 30 min in the dark, followed by microscopic observation of the LDs and autophagosomes [37 (link)]. In addition, the ultrastructure of mycelial cells was revealed by transmission electron microscopy (TEM).
Monodansylcadaverine mdc
Manufactured by Merck Group
Sourced in United States, Germany, Italy
Monodansylcadaverine (MDC) is a fluorescent compound used in various laboratory applications. It functions as a selective stain for autophagic vacuoles and can be used to monitor autophagy in cells.
Automatically generated - may contain errors
Lab products found in correlation
Acridine orange, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Beclin 1, by Cell Signaling Technology (5 mentions)
3 methyladenine 3 ma, by Merck Group (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Propidium iodide, by Merck Group (5 mentions)
N acetylcysteine nac, by Merck Group (4 mentions)
P mtor, by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Erk1 2, by Cell Signaling Technology (3 mentions)
Hoechst 33258, by Merck Group (3 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Lysotracker, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated annexin 5, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
P p70s6k, by Cell Signaling Technology (2 mentions)
101 protocols using monodansylcadaverine mdc
1
Staining Fungal Organelles for Microscopy
2
Ursolic Acid Inhibits Autophagy in Cells
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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Ursolic acid (UA) was purchased from Nanjing Zelang Plant Extract Co., Ltd. (Nanjing, China). Epirubicin was purchased from Rhawn (Shanghai, China). Autophagy inhibitor, 3-Methyladenine (3-MA), was purchased from Selleck Chemicals (Houston, TX, USA). Protease inhibitor, ECL luminescence reagent, Cell lysis buffer, Cell Counting Kit-8, Caspase3 ELISA Kit, Bradford Protein Assay Kit and autophagy agonist, rapamycin (RAPA), were obtained from Solarbio (Beijing, China). Class I PI3K, AKT, Beclin-1, LC3-II/LC3-, Atg5, Atg7 and β-Actin antibodies were purchased from CST (USA). RIPA Lysis Buffer (RIPA) was purchased from Biosharp (Guangzhou, China). Monodansylcadaverine (MDC) and Transwell chambers were purchased from Sigma (Shanghai, China). BCA Protein Assay Kit (BCA) was purchased from Bioteke (Beijing, China).
3
Lysosome and Autophagy Assays
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LysoTracker (Molecular Probes, USA) was added to cultured media for 30 min, and each well was washed three times with DMEM, and fixed with 2% paraformaldehyde for 10 min at 4°C. The red fluorescence of LysoTracker was visualized using Nikon Labphoto-2 fluorescence microscope.
Cells were incubated with 0.05 mM monodansylcadaverine (MDC) (Sigma-Aldrich, USA) at 37°C for 1 h, and the changes of fluorescence were observed by Nikon Labphoto-2 fluorescence microscope at excitation wave length 380 nm with emission filter 525 nm.
Cells were incubated with 0.05 mM monodansylcadaverine (MDC) (Sigma-Aldrich, USA) at 37°C for 1 h, and the changes of fluorescence were observed by Nikon Labphoto-2 fluorescence microscope at excitation wave length 380 nm with emission filter 525 nm.
4
Drugged AML-12 Cell Invasion Assay
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Invasion assays on an AML-12 cell line were performed in the presence of different inhibitors. AML-12 cells were pre-treated with drugs or their mock: amiloride (Sigma) at 1 mM for 30 min in culture medium, chlorpromazine CPZ (Sigma) at 10 μg/mL for 1 h in culture medium, monodansylcadaverine MDC (Sigma) at 300 μM for 1 h in DMSO, filipin (Sigma) at 5 μg/mL for 30 min in ethanol or genistein (Sigma) at 200 μM, for 30 min in DMSO. The dilution-effect of each drug was also performed. Viability of the bacteria was checked in the presence of all the dilutions of the drugs used. Bacteria (MOI = 10) diluted in the presence of the different drugs were deposited on cells for 1.5 h. Gentamicin was added for 1.5 h (using the same procedure as for the cell invasion assays). The number of internalized bacteria was determined and expressed in relation to values obtained for cells treated with control-diluted reagent arbitrarily set at 100%. Data are presented as the mean ± SEM. in duplicate and were repeated at least twice for each strain.
5
Serotonin Receptor Modulator Protocols
6
Comparative Investigation of Anti-Cancer Agents
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ALLN (#sc-221236), pifithrin-alpha (PFT-α, #sc-45050), and rapamycin (Rapa, #sc-3504A) were purchased from Santa Cruz Biotechnology. CDDP (#232120) was obtained from MERCK. 2′,7′-dichlorofluorescin diacetate (DCFDA, # D6883), monodansylcadaverine (MDC, # D4008), CQ (#C6528), and propidium iodide (PI; #P4864) were purchased from Sigma; N-Acetyl-L-cysteine (NAC, #47866) and 3-(4, 5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT, #33611) were obtained from SRL. Geneticin (G418, # 10131-035), FITC conjugated Annexin V (#A13199), Annexin V binding buffer (#V13246), and LysoTracker Green DND-26 (# L7526) were procured from Thermo Fisher Scientific. Lipofectamine 3000 was from Invitrogen (#L3000-001). The MAPK inhibitor, U0126, and gene specific primary and secondary antibodies were obtained from Cell Signaling Technology (CST, USA). pCMV-Neo-Bam p53 wt (Addgene Plasmid #16434), pCMV-Neo-Bam p53 R273H (Addgene Plasmid #16439), and pCMV-Neo-Bam Empty Vector (Addgene Plasmid # 16440 ) were a gift from Bert Vogelstein.
7
Autophagy Modulation in Cell Cultures
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All cell culture reagents were from Gibco (CA, USA). 3-methyladenine (3MA), bafilomycin A1 (Baf), rapamycin, and monodansylcadaverine (MDC) were obtained from Sigma Aldrich (St. Louis, MO, USA). LC3B, p-mTOR, mTOR, pp70S6K, p70S6K, Bax, and Bcl-2 were purchased from Cell Signaling Technology (Beverly, MA, USA). H2O2 and toluidine blue were acquired from Sangon Biotech (Shanghai, China).
8
Assessing Autophagic Activity with MDC
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Cells were sub-cultured in 6 cm-diameter Petri dishes at a quantity of 1 × 105 cells per plate and incubated in appropriate media with OA, Br-HIMOLID, or HIMOXOL for 24 h. After treatment, the cells were washed twice with PBS, incubated with 0.05 mM monodansylcadaverine (MDC) (Sigma-Aldrich, St Louis, MO, USA) in PBS at 37 °C for 30 min and then washed three times with PBS at room temperature (RT). The mean fluorescence intensities (MFIs) from intracellular MDC were measured with flow cytometry (FACScan, Becton-Dickinson, Franklin Lakes, NJ, USA) [9 (link)]). The results are representative of two independent experiments ([MFIs] ± SD).
9
Cisplatin-Resistant Ovarian Cancer Cell Lines
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The human ovarian cancer cell lines, A2780 and SKOV3, were obtained from the European Collection of Authenticated Cell Cultures (93112519, ECACC) and the American Type Culture Collection (HTB-77, ATCC), respectively and maintained in RPMI-1640 (10–040-CVR, Corning, Inc.) and McCoy 5A medium (16600-108, Gibco; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. The cisplatin-resistant cells, SKOV3/DDP and A2780/DDP, were established by exposing the cells to gradually increasing concentrations (from 0 to 25 µg/ml) of cisplatin for 6 months (data not shown). Cisplatin was purchased from Hansoh Pharmaceutical Co., Ltd. 3-Methyladenine (3-MA), rapamycin (Rapa), compound C and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich. The primary antibody to TRP14 (ab121725/dilution, 1:500) was purchased from Abcam. The primary antibodies for ATG5 (#12994/dilution, 1:1,000), Beclin1 (#3495/dilution, 1:1,000), LC3B (#3868/dilution, 1:1,000), p-5′ AMP-activated protein kinase (AMPK; #2537/dilution, 1:1,000), p-mammalian target of rapamycin (mTOR; #5536/dilution, 1:1,000), p-p70S6K (#9204/dilution, 1:1,000), AMPK (#5832/dilution, 1:1,000), mTOR (#2983/dilution, 1:1,000) and p70S6K (#2708 /dilution, 1:1,000) were obtained from Cell Signaling Technology Inc. The antibody for GAPDH (sc-47724) was purchased from Santa Cruz Biotechnology.
10
Apoptosis Signaling Pathway Assays
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Secondary mouse IgGκ BP-HRP antibody (sc-516102), mouse anti-Bax (sc-20067), mouse anti-Bcl-2 (sc-7382), mouse anti-caspase-9 (sc-56076), mouse anti-caspase-3 (sc-56053), mouse anti-MMP-9 (sc-93859), mouse anti-cytochrome C (sc-13561), mouse anti-LC3I/LC3II (sc-271625), mouse anti-p62/SQSTM1 (sc-28359), and mouse anti-β-actin (sc-69879) were purchased from Santa Cruz Biotechnology ((Santa Cruz, CA, USA). Dulbecco’s Modified Eagle Medium (DMEM) and Fetal Bovine Serum (SFB) were purchased from Gibco (GRAND Island, NE, USA). Bradford reagent was obtained from Bio-Rad. CellTiter-Glo® was purchased from Promega (WI, USA). Annexin V-FITC, camptothecin (CPT), dichlorodihydrofluorescein diacetate (H2DCFDA), Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), diphenyl-1-pyrenylphosphine (DPPP) DNase-free RNase, and LysoTracker® Red were purchased from Thermo Fisher Scientific (CA, USA). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), glutathione (GSH), acridine orange (AO), tetramethylrhodamine ethyl ester (TMRE), propidium iodide (PI), o-phthaldialdehyde (OPA), and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich (MO, USA).
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