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Antibiotic antimycotic mixed solution

Manufactured by Nacalai Tesque
Sourced in United States, Japan

Antibiotic/antimycotic mixed solution is a sterile, ready-to-use liquid that contains a combination of antibiotics and antifungal agents. It is designed for laboratory use to prevent bacterial and fungal contamination in cell culture applications.

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9 protocols using antibiotic antimycotic mixed solution

1

Cell Culture of C2C12 and HEK293T

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Mouse C2C12 myoblast and human embryonic kidney 293T (HEK293T) cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). C2C12 myoblasts and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 10% fetal bovine serum (FBS; Sigma-Aldrich, Tokyo, Japan) and an antibiotic/antimycotic mixed solution (100 U/mL penicillin G, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B) (Nacalai Tesque). Cells were cultured in a humidified atmosphere at 5% CO2 and 37 °C; the medium was replaced every two days.
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2

Cell Viability Quantification on Polymer-Coated Discs

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ECs (6 × 104) were seeded on polymer-coated polycarbonate discs (6 × 103 cells/cm2), which were placed in PMPC-coated 6 well plates (Lipidure-Coat Multi-Dish A-6MD; NOF Corporation, Tokyo, Japan). Antibiotic-Antimycotic Mixed solution (Nacalai tesque, Kyoto, Japan) was added to media at seeding to a final concentration of 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B. After 1 and 4 days of culture, attached cells were washed with phosphate-buffered saline and harvested by treatment with 0.05% trypsin-EDTA solution (Gibco/Life Technologies, Carlsbad, CA, USA). The cells were centrifuged at 450 × g for 5 min and resuspended in fresh culture medium. Aliquots of suspended cells were stained with an Acridine Orange/Propidium Iodide Viability Kit (Logos Biosystems, Annandale, VA, USA) and quantified using a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems).
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3

Isolation and Differentiation of Cardiac Fibroblasts

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Cardiac fibroblasts were isolated from the ventricle of male Wistar rats (4–5-week-old) and cultured as described previously35 (link). The isolated heart was perfused with 0.02% collagenase (Wako) by a Langendorff apparatus. Then, the ventricle was minced and suspended in Dulbecco’s modified Eagle’s medium (DMEM, Wako). After centrifugation, the suspended cells were dispersed in DMEM containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, U.S.A.) and 1% antibiotic–antimycotic mixed solution (Nacalai Tesque, Kyoto, Japan) on 2% gelatin-coated culture dish and incubated for 90 min at 37 °C in 5% CO2. After incubation, the floating cells were removed and the adhered cells were cultured in DMEM containing 10% FBS and 1% antibiotic–antimycotic mixed solution. The cells (passage 1–2) were starved for 24 h in DMEM before treatment. Differentiation of cardiac fibroblasts into myofibroblasts was induced by a stimulation of human recombinant TGF-β1 (10 ng/ml) for 48 h. Recombinant canstatin was treated 30 min before the TGF-β1 stimulation.
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4

Maintenance of Mammalian Kidney Cell Lines

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Mammalian kidney epithelial cell lines used included MDCK-NBL2 (dog), NRK-52E (rat), Vero (monkey), MDBK-NBL1 (cow), TCMK-1 (mouse), and HK-2 (human). MDCK, Vero, TCMK, and MDBK cells were maintained in Eagle’s minimum essential medium (MEM) (Sigma-Aldrich, Darmstadt, Germany) containing 10% fetal bovine serum or 10% horse serum (Nacalai Tesque, Kyoto, Japan) at 37°C and 5% CO2. NRK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, MA, United States) with 4 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, and with 5% bovine calf serum (Nacalai Tesque) at 37°C and 5% CO2. HK-2 cells were maintained in keratinocyte serum-free (KSF) medium with 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant epidermal growth factor (Gibco – Thermo Fisher Scientific, Waltham, MA, United States). All culture conditions contained a 5% antibiotic/antimycotic mixed solution (Nacalai Tesque). Cells were treated with a 0.1% trypsin – EDTA solution (Nacalai Tesque) to dislodge the cells during each passage process.
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5

Isolation and Depletion of IL-33 from Ileum Homogenate

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Ileum tissue was collected from WT or Fn14 KO mice two days after 5-FU administration, and 28 mg was minced in 1 mL of MEF culture medium (described above) with Antibiotic-Antimycotic Mixed Solution (Nacalai tesque. Kyoto, Japan). The sample was centrifuged at 300 × g for 5 min. The supernatant fraction was collected as ileum homogenate. The homogenate was mixed in a 1:9 ratio with MEF culture medium. IL-33 was depleted from the homogenate by adding 2 µg of anti-IL-33 antibody or total goat IgG (Jackson ImmunoResearch, West Grove, PA, United States) to 1 mL of homogenate and incubating for 1 hour at 4 °C. Antibodies were removed by adding 20 µL of Protein G PLUS-Agarose (resuspended volume, Santa Cruz biotechnology) and incubating for 1 h at 4 °C. After centrifugation, the supernatant fraction (i.e., the IL-33-depleted homogenate) was used for cell culture.
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6

Isolation and Culture of Neonatal Rat Cardiomyocytes

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NRCMs were isolated from neonatal Wistar rats as described previously [15 (link)]. The hearts harvested from 1–3-day-old Wistar rats were washed in PBS with 20 mM 2,3-butanedione monoxime (BDM) on ice. Then, the ventricles of the hearts were minced into small pieces and washed in wash solution (Hank’s Balanced Salt Solution with 0.08% trypsin and 20 mM BDM) for 2 h at 4 °C with stirring followed by an incubation in collagenase solution (Leibovitz’s L15 medium with 0.15% collagenase and 20 mM BDM) for 30 min at 37 °C. The suspension, tissue fragments of which were removed by filtration, was centrifuged at 100× g for 5 min at 4 °C, and the pellet was resuspended in high- glucose Dulbecco’s modified Eagle medium (DMEM; Wako, Osaka, Japan) containing 10% feral bovine serum (FBS; Gibco/Lifetechnologies, Carlsbad, CA, USA), 1% antibiotic-antimycotic mixed solution (Nacalai tesque, Kyoto, Japan) and 100 µM bromodeoxyuridine (BrdU). The cell suspension was pre-plated for 90 min twice to remove the attached non-cardiomyocytes. The non-attached cardiomyocytes were collected, seeded and cultured on culture dishes (for lucigenin assay) or coverslips coated with 1% gelatin (for DCF-DA staining or measurement of [Ca2+]i) in high-glucose DMEM containing 10% FBS, 1% antibiotic-antimycotic mixed solution and 100 µM BrdU.
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7

HLA Expression Analysis by FACS

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Reagents Abacavir sulfate (Carbosynth, Berkshire, U.K.) was dissolved in MilliQ water. Carbamazepine (Wako, Osaka, Japan) was dissolved in dimethyl sulfoxide (DMSO). For fluorescence-activated cell sorting (FACS) analysis, fluorescein isothiocyanate (FITC) anti-HLA-A,B,C (W6/32) antibody (Biolegend, San Diego, CA, U.S.A.), anti-M13 antibody (E1; Abcam, Cambridge, U.K.), anti-B17 antibody (0196HA; One LAMBDA, Canoga Park, CA, U.S.A.), and anti-HLA class I heavy chain antibody (HC10; Nordic-MUbio, Susteren, the Netherlands) were used.
Cell Culture HeLa cells, purchased from RIKEN Cell Bank (Tsukuba, Japan), were maintained in minimum essential medium (MEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, U.S.A.) plus antibiotic-antimycotic mixed solution (Nacalai Tesque) and non-essential amino acids solution (Nacalai Tesque). Cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 in air.
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8

Extraction of Colon Epithelial Tissues from Fukushima Cattle

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Colon epithelial tissues were obtained from a fetus of Japanese Black cattle (male, about 5 age in month), which was resected from euthanized parent cattle that were raised in the evacuation zone surrounding the Fukushima Daiichi Nuclear power plant accident. All procedures were authorized by the Animal Experiments and Related Activities Office at Tohoku University (Regulation No: 2014kado-037). The colon tissue was cut in parallel to intestinal tract that is 3 cm long in inside 1cm from anus. The tissue was gently washed with phosphate buffered saline (PBS) (NISSUI PHAMACEUTICAL CO., LTD., Tokyo, Japan). The epithelial layer including mucosa was scraped with a sterilized knife into a 100 mm dish coated with atelocollagen (KOKEN CO., LTD, Tokyo, Japan) and containing Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, US) and 1% Antibiotic-Antimycotic Mixed Solution (Nacalai Tesque). The dish maintained at 37°C in an atmosphere containing 5% CO2 and medium change was conducted every three days.
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9

Cell Culture Conditions Across Species

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(dog), NRK-52E (rat), Vero (monkey), MDBK-NBL1 (cow), TCMK-1 (mouse), and HK-2 (human). MDCK, Vero, TCMK, and MDBK cells were maintained in Eagle's minimum essential media (MEM) (Sigma-Aldrich, Darmstadt, Germany) containing 10% fetal bovine serum or 10% horse serum (Nacalai Tesque, Kyoto, Japan) at 37 °C and 5% CO2. NRK cells were maintained in Dulbecco's modified Eagle's media (DMEM) (Thermo Fisher Scientific, MA, USA) with 4 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, and with 5% bovine calf serum (Nacalai Tesque) at 37 °C and 5% CO2. HK-2 cells were maintained in keratinocyte serum-free (KSF) media with 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant epidermal growth factor (Gibco -Thermo Fisher Scientific, MA, USA). All culture conditions contained a 5% antibiotic / antimycotic mixed solution (Nacalai Tesque). Cells were treated with a 0.1% trypsin -EDTA solution (Nacalai Tesque) to dislodge the cells during each passage process.
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