To ablate the mycobiome in mice, animals were administered Amphotericin B (1mg/ml; MP Biomedicals, Santa Ana, CA) by oral gavage daily for five consecutive days in addition to adding Amphotericin B (0.5μg/ml) to mouse drinking water for the duration of the experiment10 (link). Controls were gavaged with PBS. Orthotopic PDA tumor cells were administered or pancreatitis was initiated 3 weeks after commencing treatment with Amphotericin B. Alternatively, mice were treated with Fluconazole (0.5mg/ml; MP Biomedicals) for 3 weeks prior to tumor implantation using the same regimen11 (link). For species-specific repopulation experiments, M. globosa (MYA-4612, 1×108 CFU/ml), S. cerevisiae (7752, 1×108 CFU/ml), C. tropicalis (MYA-3404, 1×108 CFU/ml; all ATCC Manassas, VA), Candida sp. (clinical isolate; 1×108 CFU/ml) or Aspergillus sp. (clinical isolate; 1×106 CFU/ml) were used to orally gavage mice following fungal ablation with Amphotericin B. Recipient mice were administered orthotopic PDA cells 7 days after repopulation. To assess fungal translocation to the pancreas, 108 CFU of GFP-labeled Saccharomyces cerevisiae (ATCC MYA-2011) were introduced via oral gavage, and pancreatic samples were examined at 30 minutes by flow cytometry. All experiments were approved and in compliance with the New York University School of Medicine Institutional Animal Care and Use Committee (IACUC).
Amphotericin b
Manufactured by American Type Culture Collection
Sourced in United States
Amphotericin B is an antifungal agent that is commonly used in cell culture applications. It is effective against a broad spectrum of fungal species and is often used to prevent fungal contamination in cell culture media.
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Lab products found in correlation
Penicillin, by American Type Culture Collection (7 mentions)
Streptomycin, by American Type Culture Collection (7 mentions)
Gentamicin, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (3 mentions)
Fluconazole, by MP Biomedicals (2 mentions)
M globosa mya 4612, by American Type Culture Collection (2 mentions)
S cerevisiae, by American Type Culture Collection (2 mentions)
C tropicalis mya 3404, by American Type Culture Collection (2 mentions)
Saccharomyces cerevisiae, by American Type Culture Collection (2 mentions)
Amphotericin b, by MP Biomedicals (2 mentions)
Penicillin streptomycin, by American Type Culture Collection (2 mentions)
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
Mitomycin c, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Phenol red, by American Type Culture Collection (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Glutamine, by American Type Culture Collection (1 mentions)
Mlg2908, by American Type Culture Collection (1 mentions)
Mrc 5 human lung fibroblasts, by American Type Culture Collection (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
13 protocols using amphotericin b
1
Ablating Mycobiome in Murine Pancreatic Cancer
2
Preparation of Lung Fibroblasts for Organoid Culture
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Mlg mouse lung fibroblasts ([MLg2908, CCL206], ATCC, Wesel, Germany) were maintained in DMEM/F12 medium (Life technologies, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria), penicillin/streptomycin (100 U/ml), glutamine (1%, Life Technologies #35050–061) and Amphotericin B (1×, Gibco). MRC5 human lung fibroblasts (ATCC #CCL-171) were maintained in F12 medium supplemented with 10% FBS, penicillin/streptomycin (100 U/ml), glutamine (1%) and Amphotericin B (1×). Cells were maintained at 37 °C in a humidified incubator with 5% CO2. Prior to organoid culture, Mlg or MRC5 cells at 90% confluence were proliferation-inactivated in medium containing mitomycin C (10 μg/ml, Sigma #M4287) for 2 h, followed by 3 washes in warm PBS (Life technologies) and trypsinization.
3
Fibroblast Culture and Proliferation Assay
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Human lung fibroblasts MRC5 (CCL-171; ATCC, Wesel, Germany) were cultured in Ham’s F12 medium (Life technologies, Carlsbad, United States) supplemented with 10% (v/v) fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria), 2 mM l -glutamine (Life Technologies #35050–061), 100 U/mL penicillin/streptomycin, and 1% amphotericin B (1x, Gibco). CCL-206 mouse lung fibroblasts ([MLg2908, CCL206], ATCC, Wesel, Germany) were cultured in DMEM/F12 medium supplemented with 10% FBS, penicillin/streptomycin (100 U/mL), glutamine (1%) and amphotericin B. Cells were incubated at 37°C, 5% CO2 humidified environment. MRC5 or CCL206 fibroblasts were starved once grown to 80% confluence in the 6-well culture plates. The starvation medium contains the same components as culture medium described above, but with only 0.5% FBS. After 24 h starvation, cells were then incubated with either vehicle, TGF-β1 and/or investigational compounds (Table 1 ) in serum deprivation medium for 48 h. MRC5 fibroblasts were collected for gene expression analysis. CCL206 fibroblasts were washed three times with warm PBS and proliferation-inactivated by incubation in mitomycin C (10 μg/ml, Sigma #M4287) for 2 h, followed by three washes in warm PBS and trypsinization prior to mixing with epithelial cells, as described previously (Ng-Blichfeldt et al., 2018 (link); Ng-Blichfeldt et al., 2019 (link)).
4
Culturing Mammary Cell Lines
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MCF-7 cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM (Genesee Scientific) supplemented with 10% FBS (Gemini Bio-Products), 1% non-essential amino acids (Gemini Bio-Products), and 1% antibiotic/antimycotic solution (Gemini Bio-Products) containing 100 IU/ml penicillin, 100 µg/ml streptomycin and 25 µg/ml amphotericin B. Primary human mammary epithelial cells (HMECs) were obtained from ATCC, and cultured in Mammary Epithelial Cell Basal Medium (ATCC) supplemented with a Mammary Epithelial Cell Growth kit (ATCC) containing 5 µg/ml hH-insulin, 6 mM L-glutamine, 0.5 µM epinephrine, 5 µg/ml apo-transferrin, 5 ng/ml rH-TGF-α, 0.4% ExtractP and 100 ng/ml hydrocortisone hemisuccinate. MCF-10A cells were obtained from ATCC and cultured in DMEM/Ham's F12 media supplemented with 5% Equine Serum (Gemini Bio-Products), 20 ng/ml epidermal growth factor (Sigma-Aldrich; Merck KGaA), 10 µg/ml insulin (Sigma-Aldrich; Merck KGaA), 0.5 mg/ml hydrocortisone (Sigma-Aldrich; Merck KGaA), 100 ng/ml cholera toxin (Sigma-Aldrich; Merck KGaA) and 1% antibiotic/antimycotic solution. All cells were maintained in 5% CO2 at 37°C in a humidified incubator.
5
Maintenance of Human Aortic SMCs
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Primary human vascular aortic smooth muscle cells (HAoSMCs) (American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in a humidified atmosphere at 37℃ with 5% CO2, and cultured in VSMC basal medium supplemented with 5 ng/mL rhFGF-basic, 5 µg/mL rhInsulin, 5 ng/mL rhEGF, 10 mM L-glutamine, 50 µg/mL ascorbic acid, 5% fetal bovine serum, 10 µg/mL gentamicin, 10 Units/mL penicillin, 10 µg/mL streptomycin, 0.28 µg/mL amphotericin B, and 33 µM phenol red (ATCC, Manassas, VA, USA).
6
Ablating Mycobiome in Murine Pancreatic Cancer
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To ablate the mycobiome in mice, animals were administered Amphotericin B (1mg/ml; MP Biomedicals, Santa Ana, CA) by oral gavage daily for five consecutive days in addition to adding Amphotericin B (0.5μg/ml) to mouse drinking water for the duration of the experiment10 (link). Controls were gavaged with PBS. Orthotopic PDA tumor cells were administered or pancreatitis was initiated 3 weeks after commencing treatment with Amphotericin B. Alternatively, mice were treated with Fluconazole (0.5mg/ml; MP Biomedicals) for 3 weeks prior to tumor implantation using the same regimen11 (link). For species-specific repopulation experiments, M. globosa (MYA-4612, 1×108 CFU/ml), S. cerevisiae (7752, 1×108 CFU/ml), C. tropicalis (MYA-3404, 1×108 CFU/ml; all ATCC Manassas, VA), Candida sp. (clinical isolate; 1×108 CFU/ml) or Aspergillus sp. (clinical isolate; 1×106 CFU/ml) were used to orally gavage mice following fungal ablation with Amphotericin B. Recipient mice were administered orthotopic PDA cells 7 days after repopulation. To assess fungal translocation to the pancreas, 108 CFU of GFP-labeled Saccharomyces cerevisiae (ATCC MYA-2011) were introduced via oral gavage, and pancreatic samples were examined at 30 minutes by flow cytometry. All experiments were approved and in compliance with the New York University School of Medicine Institutional Animal Care and Use Committee (IACUC).
7
Hyperglycemic hASMC Culture and Treatment
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Primary human aortic smooth muscle cells (hASMCs) (ATCC PCS-100-012, American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in a humidified atmosphere of 37 °C, with 5% CO2 in VSMC basal medium (without glucose and phenol red) (ATCC® PCS-100-030™) supplemented with recombinant human basic fibroblast growth factor (5 ng/mL), rhInsulin (5 µg/mL), recombinant human epidermal growth factor (5 ng/mL), L-glutamine (10 mM), ascorbic acid (50 µg/mL), fetal bovine serum (5%), gentamicin (10 µg/mL), penicillin (10 Units/mL), streptomycin (10 µg/mL), amphotericin B (0.28 µg/mL), and phenol red (33 µM) (ATCC). To induce clinically hyperglycemic condition, we stimulated the cells with 25 mM (450 mg/dL) of glucose. Based on our previous study for cell viability [27 (link)], we used 1 and 10 μg/mL concentration of C. turczaninowii extract in this study.
8
Culturing Primary Keratinocytes and HaCaT Cells
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Primary NHEK cells from neonatal foreskin (PCS-200-010) were purchased from American Type Culture Collection (ATCC, Manassas, VA). The NHEK cells were cultured in the Dermal Cell Basal Medium (ATCC, Manassas, VA) supplemented with the Keratinocyte Growth Kit (ATCC, Manassas, VA) and antibiotics (10 µg/mL of gentamicin, 0.25 µg/mL of amphotericin B, 10 U/mL of penicillin, 10 µg/mL of streptomycin, 25 ng/mL of amphotericin B; ATCC, Manassas, VA) according to the manufacturer's instructions. Immortalized nontumorigenic human keratinocytes, HaCaT cells [15] (link), were kindly provided by Dr. Norbert E. Fusenig (German Cancer Research Center, Heidelberg, Germany). The HaCaT cells were cultured in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Laboratories Inc., Logan, UT) and antibiotics (100 U/mL of penicillin, 100 µg/mL of streptomycin, and 2 mM L-glutamine; Gibco BRL, Grand Island, NY). Both the NHEK and HaCaT cells were incubated at 37°C in humidified air containing 5% CO2.
9
Kidney Proximal Tubule Cell Culture
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Human kidney cortex proximal tubule epithelial cells (HK-2, ATCC CRL-2190, ATCC, United Kingdom) were cultured according to the instructions from the manufacturer in keratinocyte serum-free medium, with added bovine pituitary extract (0.05 mg/mL) and epidermal growth factor (5 ng/mL) (all from Invitrogen, United Kingdom). Human primary renal proximal tubule epithelial cells (RPTEC, ATCC PCS-400-010, ATCC) were cultured according to the instructions from the manufacturer. RPTE cells were grown in renal epithelial cell basal media, supplemented with renal epithelial cells growth kit components and antimicrobials: gentamicin (10 μg/mL), amphotericin B (0.275 μg/mL), penicillin (10 units/mL) and streptomycin (10 μg/mL) (all from ATCC). Cell cultures were incubated at 37 °C in the presence of 5% CO2. When cells reached 80–90% confluence, heme (0.5–30 μM, from a freshly prepared 10 mM stock solution), or a mixture of ammonium iron(III) sulfate dodecahydrate, hydrogen peroxide, and ascorbic acid (generating hydroxyl radicals via the Fenton reaction), with or without addition of either Hp (10 μM) or rA1M (10 or 12 μM) or rA1M only, was added, and cells were incubated for up to 24 h. For details on exposure concentrations and time, see the respective figure.
10
Hematological Cancer Cell Line Panel
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