C. albicans DAY185 overnight cultures were diluted in half-strength potato dextrose broth (PDB; Becton Dickinson, Scoresby, Australia) to an OD600 of 0.2 and grown for a further three hours (30 °C, 250 rpm). Cells were then diluted to an OD600 of 0.3 with half-strength PDB. For time course experiments, 300 µL aliquots (in 1.5 mL microcentrifuge tubes) were pre-treated with 5 µM propidium iodide (PI; Sigma, St Louis, MO, USA) for 10 min prior to addition of BODIPY- labelled NaD1. For CCCP and latrunculin A experiments, yeast cells were pre-treated with 50 µM CCCP (Sigma) or 100 µM latrunculin A (AdipoGen, Liestal, Switzerland) for 2 h (30 °C, 250 rpm) before addition of 5 µM PI. BODIPY-NaD1 (10 µM) was then added and cells were monitored using a Zeiss LSM510/ConfoCor confocal with images taken every 5 sec. BODIPY was excited at 488 nm (Argon laser) and emission was detected at 505 to 530 nm. PI was excited at 561 nm (DPSS laser) and fluorescence was monitored at 575 to 615 nm. Images were captured using Zen2009 (Zeiss, Oberkochen, Germany) software and analysed using FIJI (Bethesda, Rockville, MD, USA) [29 (link)]. Brightness and contrast for Figure 1 , Figure 3 and Figure 4 were adjusted using the auto-brightness/contrast function of FIJI.
Latrunculin a
Manufactured by Adipogen
Sourced in Switzerland, United Kingdom, Austria
Latrunculin A is a natural product isolated from the Red Sea sponge Negombata magnifica. It is a potent inhibitor of actin polymerization and is commonly used as a research tool to investigate actin dynamics in cells.
Automatically generated - may contain errors
Lab products found in correlation
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Zen 2009, by Zeiss (1 mentions)
Latrunculin a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Molecular weight marker for western blotting, by GenDEPOT (1 mentions)
Kn 93, by Merck Group (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Mag fluo 4 am, by Thermo Fisher Scientific (1 mentions)
N benzyl p toluene sulfonamide bts, by Bio-Techne (1 mentions)
Insulin, by Merck Group (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Gefitinib, by Cayman Chemical (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Pf562271, by Adipogen (1 mentions)
Zstk474, by Cell Signaling Technology (1 mentions)
Latrunculin b, by Enzo Life Sciences (1 mentions)
Ly294002, by Cayman Chemical (1 mentions)
Myriocin, by Merck Group (1 mentions)
Nourseothricin, by Jena Biosciences (1 mentions)
Nocodazole, by Merck Group (1 mentions)
5 protocols using latrunculin a
1
Multifunctional Fluorescent Assay for Candida
2
Reagents for Calcium Signaling Assays
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N-benzyl-p-toluene sulfonamide (BTS) was purchased from Tocris Biosciences (Bristol/UK, England), latrunculin A from AdipoGen (San Diego/California, United States), and 4-chloro-m-cresol (4CmC) from Pfaltz & Bauer (West Chester/Pennsylvania, United States). The calcium dyes fura-2 AM and mag-fluo-4 AM were purchased from Invitrogen (Grand Island/New York, United States). Molecular weight marker for western blotting was purchased from GenDEPOT (Barker/TX, United States). Every other reagent including Insulin, KN-92, KN-93, and the myristoylated autocamtide 2-related inhibitory peptide (AIP) were purchased from Sigma-Aldrich (St. Louis, United States).
3
Preparation of Pharmacological Reagents for Cell-Based Assays
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Stocks of 50 mM LY294002 (Cayman, #70920), 5 mM Latrunculin A (AdipoGen, #AGCN20027C100), 25 mM Latrunculin B (Enzo, #BML-T110-0001), 10 mM MEK inhibitor PD325901 (Calbiochem, #444966), 10 mM FAK inhibitor PF-573228 (Cayman, #14924), 10 mM FAK inhibitor PF-562271 (AdipoGen, SYN-1064), 10 mM ZSTK474 (Cell Signaling, #13213), 1 mM Gefitinib (Cayman, #13166), and 10 mM Rapamycin (Cayman, #13346) were prepared by dissolving the chemicals in DMSO. The stocks were diluted to the indicated final concentrations in culture medium. The EGF stock solution was prepared by dissolving EGF (Sigma, #E9644) in 10 mM acetic acid to a final concentration of 1 mg/ml. All drug stocks were stored at −20 °C.
4
Yeast Growth Protocols for Cell Cycle Studies
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Yeast strains were grown in synthetic media supplemented with 2% glucose and 20 mg/L adenine [20 ]. Methionine-free media were used to grow cells that carry the MET3-CDC20 cassette. Unless otherwise indicated, all procedures were conducted at 23°C.
Standard chemicals were from Sigma/Aldrich. Other chemicals were obtained from the following sources: alpha factor peptide (Cleveland Clinic Core Laboratory, Cleveland, OH), DiOC6 (Thermo Fisher Scientific), FM4-64 (Thermo Fisher Scientific), G418 (Amresco), latrunculin A (AdipoGen Life Sciences), myriocin (Sigma-Aldrich), nocodazole (Sigma/Aldrich), nourseothricin (Jena Bioscience), rhodamine-Concanavalin A (Vector Laboratories).
Standard chemicals were from Sigma/Aldrich. Other chemicals were obtained from the following sources: alpha factor peptide (Cleveland Clinic Core Laboratory, Cleveland, OH), DiOC6 (Thermo Fisher Scientific), FM4-64 (Thermo Fisher Scientific), G418 (Amresco), latrunculin A (AdipoGen Life Sciences), myriocin (Sigma-Aldrich), nocodazole (Sigma/Aldrich), nourseothricin (Jena Bioscience), rhodamine-Concanavalin A (Vector Laboratories).
5
Fungal Growth Inhibition Assays
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Fungal growth inhibition assays were performed essentially as described in [30 (link)]. Overnight cultures of C. albicans cells were diluted to 5000 cells/mL with half-strength PDB. For growth inhibition assays in the presence of latrunculin A, 80 µL of diluted C. albicans DAY185 cells were added to 10 µL of latrunculin A and 10 µL of NaD1 in a 96 well microtiter plate (Greiner, Kremsmünster, Austria) to final concentrations of 2.5, 3, 4 and 4.5 µM NaD1 and 0 or 20 μM latrunculin A (AdipoGen). For Brefeldin A and nocodazole assays, 80 µL of diluted C. albicans DAY185 cells was added to 10 µL of inhibitor and 10 µL of NaD1 in a microtiter plate. Brefeldin A (Sigma) and nocodazole where serially diluted (two-fold) down the plate with top final concentrations of 40 µM and 20 µM respectively. NaD1 was added to all wells at a final concentration of 2.5 µM. No-inhibitor controls were also included. For testing the antifungal activity of NaD1 against the C. albicans ESCRT mutants, 80 µL of diluted cells (DAY286 wild type and mutants) were added to 20 µL of NaD1 serially diluted from a top final concentration of 10 µM. All plates were incubated at 30 °C overnight without shaking. Growth of cells was monitored by measuring absorbance at 595 nm in a SpectraMAX M5e plate reader (Molecular Devices, San Jose, CA, USA). Measurements were taken at t = 0 and t = 24 h.
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