Amphotericin b

Manufactured by Avantor

Sourced in United States, Macao

Amphotericin B is an antifungal agent used in laboratory settings. It is a polyene macrolide antibiotic that acts by binding to ergosterol, a component of fungal cell membranes, leading to increased permeability and cell death. Amphotericin B is commonly used for the treatment of serious fungal infections.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Vancomycin, by Avantor (4 mentions) Penicillin, by Thermo Fisher Scientific (4 mentions) Streptomycin, by Thermo Fisher Scientific (4 mentions) Trimethoprim, by Merck Group (3 mentions) Polymyxin b, by Merck Group (3 mentions) Brain heart infusion broth, by BD (3 mentions) β cyclodextrin, by Merck Group (2 mentions) Brucella broth, by Neogen (2 mentions) Cefsulodin, by Merck Group (2 mentions) Vancomycin, by Thermo Fisher Scientific (2 mentions) Anoxomat gas evacuation and replacement system, by Advanced Instruments (2 mentions) Kanamycin, by Thermo Fisher Scientific (2 mentions) Ccl 33, by American Type Culture Collection (2 mentions) Crl 2935, by American Type Culture Collection (2 mentions) Penicillin g, by Avantor (2 mentions) Crl 3216, by American Type Culture Collection (2 mentions) Opti mem, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions) L glutamine, by Merck Group (2 mentions)

22 protocols using amphotericin b

Culturing HeLa Cells with Chloroquine and Isolating Murine Hepatocytes

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HeLa cells (European Cell Culture Collection) were cultured in minimum essential medium with Earle’s salts (MEM EBS; BioConcept, 1‐31F01‐I), supplemented with 10% FCS (GE Healthcare), 100 U penicillin (BioConcept), 100 μg/ml streptomycin (BioConcept), and 2 mM L-glutamine (BioConcept). Cells were cultured at 37 °C and 5% CO₂ and split using Accutase (Innovative Cell Technologies) diluted 1:2 in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4). Chloroquine treatment was performed in MEM complete supplemented with 10 μM Chloroquine (Sigma-Aldrich C6628).
Primary murine hepatocytes were isolated and cultured as described elsewhere^{3 (link)}. For infection of cells, salivary glands of infected Anopheles stephensi mosquitoes from 16–26 days after blood feeds were isolated and homogenized to release sporozoites. Sporozoites were incubated with cells in medium containing 25 μg/ml Amphotericin B (Amresco E437) for 2 h. Subsequently, cells were rinsed and incubated in MEM EBS complete containing 2.5 μg/ml Amphotericin B.

Niklaus L., Agop-Nersesian C., Schmuckli-Maurer J., Wacker R., Grünig V, & Heussler V.T. (2019). Deciphering host lysosome-mediated elimination of Plasmodium berghei liver stage parasites. Scientific Reports, 9, 7967.

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Antimicrobial Compound Preparation

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Fluconazole (J&K, Shanghai, China) and Amphotericin B (AMRESCO, Solon, OH, USA) were dissolved with dimethyl sulfoxide (DMSO, Merck-China, Chengdu, China) and stored at −20 °C until use. Vancomycin hydrochloride (Solarbio, Beijing, China) and methicillin (APExbio, Shanghai, China) were dissolved with deionized water and stored at −20 °C until use.

Hu Y., Niu Y., Ye X., Zhu C., Tong T., Zhou Y., Zhou X., Cheng L, & Ren B. (2021). Staphylococcus aureus Synergized with Candida albicans to Increase the Pathogenesis and Drug Resistance in Cutaneous Abscess and Peritonitis Murine Models. Pathogens, 10(8), 1036.

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Cultivation and Maintenance of H. pylori

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All H. pylori strains were maintained at −80°C in brain heart infusion broth (Becton Dickinson) supplemented with 10% fetal bovine serum (FBS) and 20% glycerol and were cultivated on horse blood agar (HBA) medium containing 4% columbia agar base (Neogen Corp), 5% defibrinated horse blood (HemoStat Laboratories, Dixon, CA), 0.2% β-cyclodextrin (Sigma), 10 µg/mL of vancomycin (Amresco), 2.5 U/mL of polymyxin B (Sigma), 5 µg/mL of trimethoprim (Sigma), and 5 µg/mL of amphotericin B (Amresco). Where required, 5% sucrose was added to HBA for selection of sucrose sensitive strains. Liquid growth of H. pylori was performed in brucella broth (Neogen Corp) with 10% FBS and 10µg/mL of vancomycin. All H. pylori cultures were grown under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) at 37°C with 100rpm shaking for liquid cultures. H. pylori strain G27 was used for all experiments^{12 (link)}. Escherichia coli Top10 cells were either grown on LB agar or in LB liquid medium with shaking at 225rpm. Kanamycin (25µg/mL) and ampicillin (100µg/mL) were used for bacterial selection.

Johnson R.C., Hu H.Q., Merrell D.S, & Maroney M.J. (2015). Dynamic HypA zinc site is essential for acid viability and proper urease maturation in Helicobacter pylori. Metallomics : integrated biometal science, 7(4), 674-682.

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Chlamydial Isolation from Animal Swabs

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Human Epidermoid Carcinoma-2 cells (Hep-2 cells) were grown in Iscove’s Modified Dulbecco’s Medium (IMDM, Life Technology, USA) with 10% fetal bovine serum (Life Technology, USA) and amphotericin B (250 μg/ml, Amresco, USA) at 37 °C with 5% CO₂ in a humidified cabinet for 24–48 h. Swabs in 200 μl SPG buffer from animals that were C. gallinacea-positive were transferred for chlamydial isolation into sterile tubes and vortexed with sterile magnetic beads for 3 min. After centrifugation at 1,250 g for 5 min, supernatants were transferred for incubation into 25 cm² cell culture flasks with 7.6 ml IMDM and 0.4 ml antibiotics dissolved in IMDM (vancomycin 100 μg/ml, streptomycin 100 μg/ml, kanamycin 100 μg/ml, amphotericin B 3.75 μg/ml and gentamycin 10 μg/ml).

Guo W., Li J., Kaltenboeck B., Gong J., Fan W, & Wang C. (2016). Chlamydia gallinacea, not C. psittaci, is the endemic chlamydial species in chicken (Gallus gallus). Scientific Reports, 6, 19638.

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Cultivation and Storage of Helicobacter pylori Strains

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The strains used in this study are listed in Table 1; 26695, J99, and G27 are common laboratory strains; 7.13 and SS1 are strains used in animal infection models; USU103 and UA1182 are multi-drug resistant strains. Strains were grown on horse blood agar (HBA) composed of 4% Columbia agar (Neogen Corporation), 5% defibrinated horse blood (HemoStat Laboratories, Dixon, CA), 2 mg/mL cyclodextrin (Sigma-Aldrich), and an antibiotic-antifungal cocktail composed of 10 μg/mL vancomycin (Amresco), 5 μg/mL cefsulodin (Sigma-Aldrich), 2.5 U/mL polymyxin B (Sigma-Aldrich), 5 μg/mL trimethoprim (Sigma-Aldrich), and 8 μg/mL amphotericin B (Amresco), or in liquid brucella broth (BB) (Neogen Corporation) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 10 μg/mL vancomycin. Freezing media, consisting of brain heart infusion broth (BD Biosciences) containing 10% FBS and 20% glycerol (EMD Chemicals, Inc.), was used for the creation and storage of H. pylori −80°C stock cultures.
H. pylori strains were grown as previously described (Carpenter et al., 2015 (link)). Briefly, strains were cultured in gas evacuation jars at 37°C under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂); these conditions were generated with an Anoxomat gas evacuation and replacement system (Advanced Instruments, Inc.). Liquid cultures were grown shaking at 110 RPM.

Wylie M.R., Windham I.H., Blum F.C., Wu H, & Merrell D.S. (2021). In vitro antibacterial activity of nimbolide against Helicobacter pylori. Journal of ethnopharmacology, 285, 114828.

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Antifungal Susceptibility of Blastomyces

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Clinical isolates of Blastomyces spp collected at the University of Wisconsin Hospital between 2004 and 2017 were tested for antifungal susceptibility retrospectively. They were cultured at 35°C for 10–15 days on potato dextrose agar (Oxoid, Hampshire, England) to induce conidial formation. Conidia were then suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO) at 1.0 × 10⁵ conidia/mL and 200 µL was added to 96-well plates containing the antifungals. Five antifungals were tested: itraconazole, voriconazole, and isavuconazole (Santa Cruz Biotechnology, Santa Cruz, CA), posaconazole (Selleck Chemicals, Houston, TX), and amphotericin B (AMRESCO, Solon, OH). Stock solutions were prepared in dimethyl sulfoxide (Sigma-Aldrich) except for amphotericin B, which was in sterile water. Drugs were diluted into RPMI medium via serial doubling dilutions, with concentrations ranging from 0.015 to 8.0 µg/mL except for itraconazole, which had concentrations ranging from 0.13 to 64 µg/mL.

Scolarici M.J., King C., Sterkel A., Smith J., Gauthier G, & Saddler C. (2022). The Role of Isavuconazonium Sulphate for the Treatment of Blastomycosis: A Case Series and Antifungal Susceptibility. Open Forum Infectious Diseases, 9(7), ofac220.

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Culturing and Maintaining Helicobacter pylori

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Strains and plasmids used in this study are listed in Table 1. Unless noted otherwise, H. pylori strains were grown as previously described (Carpenter et al., 2015 (link)). Briefly, H. pylori stock cultures were maintained at −80°C in H. pylori freezing media [brain heart infusion broth (BD Biosciences) containing 10% fetal bovine serum (FBS) and 20% glycerol (EMD chemicals, Inc)]. Freezer stocks were plated on horse blood agar (HBA) plates comprised of 4% Columbia agar (Neogean Corporation), 5% defibrinated horse blood (HemoStat Laboratoris, Dixon, CA), 2 mg/mL β-cyclodextran (Sigma), and an antibiotic/antifungal cocktail [10 μg/ml vancomycin (Amresco), 5 μg/ml cefsulodin (Sigma), 2.5 U/ml polymyxin B (Sigma), 5 μg/ml trimethoprim (Sigma), and 8 μg/ml amphotericin B (Amresco)]. Following growth on HBA plates, H. pylori strains were grown in liquid culture. Liquid media consisted of brucella broth (Neogen Corporation) supplemented with 10% FBS (Gibco) and 10 ug/mL vancomycin. Where indicated, 25 μg/mL Kanamycin (Kan²⁵) was used to supplement the media. All cultures were grown at 37°C, in gas evacuation jars, under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) generated with an Anoxomat gas evacuation and replacement system (Advanced Instruments, Inc.); in addition, liquid cultures were grown shaking at 100 rpm.

Servetas S.L., Doster R.S., Kim A., Windham I.H., Cha J.H., Gaddy J.A, & Merrell D.S. (2018). ArsRS-Dependent Regulation of homB Contributes to Helicobacter pylori Biofilm Formation. Frontiers in Microbiology, 9, 1497.

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Lifespan Assay of C. elegans Exposed to PA

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To a 25 mL Erlenmeyer flask, was added 10 mL of synchronized L1 worms at concentration of 300 worms/mL, 25 μL of food substrate (to give an OD₆₀₀ = 2.5), 200 μL of a 100 × penicillin/streptomycin (Amresco, Solon, OH, USA) solution in S-media, and 10 μL of a 5 mg/mL amphotericin B (Amresco, Solon, OH, USA) solution in S-media. The resulting worm suspension (100 μL) was added to each well of a 96-well plate, the plates were wrapped with paraffin to prevent evaporation, and then incubated at 20 °C on an orbital shaker at a speed of 110 rpm. At 1 day of age, we added 100 μL of a PA stock solution (24 μM of PA with 0.16% DMSO in S-media) or DMSO vehicle to each well. We used 8 replicates or 8 wells for each cohort. At 2.5 days of age, to each well, we added 10 μL of a 4.2 mM solution of FUdR in S-media. Beginning at 5 days of age, the worms were exposed to LWL (660 nm at 0.25 W/m², 5 h per day light and 19 hours darkness) or kept in the dark by covering the plate with aluminum foil. Worms were counted and scored dead or alive every 2 or 3 days until all worms were dead.

Zhang D., Robinson K., Mihai D.M, & Washington I. (2016). Sequestration of ubiquitous dietary derived pigments enables mitochondrial light sensing. Scientific Reports, 6, 34320.

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Susceptibility Testing of Candida albicans

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Candida albicans 3153A is wildtype strained. The FLO8 plasmid with GFP was purchased in BioSune Biology Company, Shanghai, China. Stock cultures of Candida albicans strains were routinely cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) solid medium containing 1.5% agar at 37 °C for 24 to 48 h. Candida albicans Yeast inocula cells were prepared by transferring a single colony into YPD medium with overnight incubation at 37 °C in an incubator shaker at about 100 rpm. Cells were harvested by centrifugation at 6,000 g for 3 min and washed twice in sterile phosphatebuffered saline (PBS [pH 7.2 to 7.4]). Then the cells were resuspended in RPMI 1640 medium (Gibco) with L-glutamine, buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS [Sigma-Aldrich]) and adjusted to the desired density of 2 × 10³~1 × 10⁴ ^{42 (link)}. CLSI M27-A3 microdilution methodology was used to test in vitro susceptibility to amphotericin B(Amresco)^{43 (link)}. LIVE-DEAD^® Funga Light yeast viability kit was purchased from Invitrogen. The link for 50,520 different small molecule compounds is: http://www.cncl.org.cn/.

Qiang L., Guo J., Han Y., Jiang J., Su X., Liu H., Qi Q, & Han L. (2019). A novel anti Candida albicans drug screening system based on high-throughput microfluidic chips. Scientific Reports, 9, 8087.

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Culturing and Maintaining Helicobacter pylori

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Helicobacter pylori strains were maintained at - 80 °C in brain heart infusion (BHI) broth (BD) supplemented with 20% (v/v) glycerol (CalBioChem) and 10% (v/v) fetal bovine serum (FBS, Gibco). Growth on plates was achieved on 4.4% (w/v) Columbia agar (Acumedia) supplemented with 5% (v/v) horse blood (HemoStat), 0.2% (w/v) β-cyclodextrin (Sigma), 10 μg mL⁻¹ vancomycin (Amresco), 2.5 U mL⁻¹ polymyxin B sulfate (Sigma), and 8 μg mL⁻¹ amphotericin B (Amresco). Liquid growth was accomplished in Brucella broth (Acumedia) supplemented with 10% (v/v) FBS and 10 μg mL⁻¹ vancomycin, with shaking at 110 RPM. Growth on plates or in liquid was performed at 37 °C, under microaerobic conditions (10% CO₂, 5% O₂, and N₂ as balance) achieved using an Anoxomat (Advanced Instruments Inc). Where appropriate, selection was performed with 5% sucrose or 25 μg mL⁻¹ kanamycin (Gibco).

Spronk C.A., Żerko S., Górka M., Koźmiński W., Bardiaux B., Zambelli B., Musiani F., Piccioli M., Basak P., Blum F.C., Johnson R.C., Hu H., Merrell D.S., Maroney M, & Ciurli S. (2018). Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 23(8), 1309-1330.

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