Antibiotic antimycotic mix

Manufactured by Merck Group

Sourced in United States, Germany

Antibiotic/antimycotic mix is a sterile solution containing a combination of antibiotics and antifungal agents. It is designed for use in cell culture applications to prevent bacterial and fungal contamination.

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Antibiotic/antimycotic (245 citations) Antibiotics/antimycotics (28 citations) Antimycotics (7 citations) Antibiotic mix (6 citations) Antibiotics mix (4 citations)

20 protocols using antibiotic antimycotic mix

Cell culture protocols for comparative studies

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Chinese hamster ovary cells (CHOwt) and CHO NPC1-null cells (kindly provided by Dr. Daniel S. Ory, Washington University School of Medicine, USA) were maintained in DMEM/F12 medium (1:1) supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine and antibiotic/antimycotic mix (all from Sigma-Aldrich) [16 (link)].
Human neuroblastoma cells (SH-SY5Y) (kindly provided by Dr. Stefan Lichtenthaler, German Center for Neurodegenerative Diseases, Germany) were maintained in DMEM/F12 medium (1:1) supplemented with 15% Fetal Bovine Serum, 2 mM L-glutamine, antibiotic/antimycotic mix (all from Sigma-Aldrich) and 1% non-essential amino acids (Gibco) [17 (link)].
Wild type mouse embryonic fibroblasts (MEFwt), cathepsin B/L knockout (KO) MEFs (CtsB^-/-, CtsL^-/-, CtsB^-/-L^-/-) (kindly provided by Dr. Oliver Schilling, Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany) and cathepsin D KO MEFs (CtsD^-/-) (kindly provided by Dr. Paul Saftig, Institute of Biochemistry, Kiel, Germany) were maintained in DMEM GlutaMAX medium (Gibco) supplemented with 10% Fetal Bovine Serum and antibiotic/antimycotic mix (all from Sigma-Aldrich) [18 (link);19 (link)].

Cermak S., Kosicek M., Mladenovic-Djordjevic A., Smiljanic K., Kanazir S, & Hecimovic S. (2016). Loss of Cathepsin B and L Leads to Lysosomal Dysfunction, NPC-Like Cholesterol Sequestration and Accumulation of the Key Alzheimer's Proteins. PLoS ONE, 11(11), e0167428.

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Culturing and Analyzing Tetrahymena Strains

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The wild-type CU428.2 and B2086.2 Tetrahymena strains were obtained from the Tetrahymena Stock Center (Cornell University, Ithaca, NY, USA). Wild-type and other motile strains were grown in the SPP (Super Proteose Peptone) medium^{50 (link)} with the antibiotic–antimycotic mix at 1:100 (Sigma-Aldrich, St-Louis, MO, USA). Mutants with greatly reduced motility were grown in MEPP (Modified Enriched Proteose Peptone) medium, on which cells take up nutrients without using oral cilia^{51 (link)}, supplemented with the antibiotic–antimycotic mix at 1:30 (Sigma-Aldrich, St-Louis, MO, USA). Cells were grown with moderate shaking (80 rpm) at 30 °C. Cell swimming and cilia beating patterns were analyzed as previously described^{52 (link),53 (link)}. To induce protein overexpression, cells were cultured in media with 2.5 µg/ml CdCl₂ for 16–18 h.

Joachimiak E., Osinka A., Farahat H., Świderska B., Sitkiewicz E., Poprzeczko M., Fabczak H, & Wloga D. (2021). Composition and function of the C1b/C1f region in the ciliary central apparatus. Scientific Reports, 11, 11760.

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Cell viability assessment using MTT assay

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HEK293 FT wild-type and APEX1-KO cells were plated into 96-well plates at a density of 1.3 × 10⁴ cells/well in DMEM with 10% fetal bovine serum (FBS) and antibiotic/antimycotic mix (Sigma, A5955-100, St. Louis, MI, USA). CH12F3 wild-type and APEX1-KO cells were collected by centrifugation and resuspended in phenol red-free RPMI-1640 with L-glutamine, 10% FBS, and antibiotic/antimycotic mix (Sigma, A5955-100, St. Louis, MI, USA) and then plated on 96-well plates at a density of 2.2 × 10⁴ cells/well. The plates were incubated for 24 h at 37 °C in a 5% CO₂ humidified incubator. Immediately prior to use, the Ape1 inhibitors and methylmethane sulfonate (MMS) were diluted with a culture medium containing 3% FBS. For both HEK293 FT and CH12F3 cells, the FBS in the medium was reduced from 10% to 3% before the addition of inhibitors or MMS. After 24 h of treatment, MTT (3-(4,5-dethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was then added at a concentration of 0.5 mg/mL, followed by a 3-h incubation. Stop solution (1% SDS and 0.01 M HCl) was used to dissolve formazan crystals, and the absorbance was measured at 590 nm.

Xue Z, & Demple B. (2022). Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression. Antioxidants, 11(9), 1817.

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Isolation of Pancreatic Cancer Cell-Derived EVs

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We used PDAC cell lines (derived from primary tumors) Panc02.03 (ATCC® CRL-2553), Panc08.13 (ATCC® CRL-2551), Panc10.05 (ATCC® CRL-2547) and Panc-1 (ATCC® CRL-1469). Panc02.03, Panc08.13 and Panc10.05 were cultured in RPMI-1640 (Gibco) supplemented with 15% FBS (Gibco), cyprofloxacine, antibiotic/antimycotic mix, glutamine (Sigma) and 10 U/ml human recombinant insulin (Santa-Cruz). Panc-1 cells were cultured in DMEM with 10% FBS (Gibco) and antibiotics. The control immortalized Human Pancreatic Duct Epithelial Cell Line (H6c7, HPDEC, Kerafast) was maintained in keratinocyte serum-free medium that contained EGF and bovine pituitary extract (Thermo Fisher, 17005042), antibiotic/antimycotic mix and glutamine (Sigma). Cells were tested for mycoplasma contamination with Hoechst staining and only negative cultures < 9 passage numbers were used in our experiments. Cells were washed three times with phosphate buffered saline (PBS) 2 days after culturing them in serum-free medium, the medium was then replaced and EVs were collected after 2 days. For 3D cultures, cells were removed from culture dishes (Eppendorf) with TrypLE (Gibco), embedded into Matrigel with 20,000 cells/droplet (25 μl) and cultured in 2.5% EV-free FBS (exosome depleted One-Shot FBS, Gibco) prior change to serum-free medium 2 days before starting EV collection.

Zeöld A., Sándor G.O., Kiss A., Soós A.Á., Tölgyes T., Bursics A., Szűcs Á., Harsányi L., Kittel Á., Gézsi A., Buzás E.I, & Wiener Z. (2020). Shared extracellular vesicle miRNA profiles of matched ductal pancreatic adenocarcinoma organoids and blood plasma samples show the power of organoid technology. Cellular and Molecular Life Sciences, 78(6), 3005-3020.

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Leptin and Sirtinol Treatment of SH-SY5Y Cells

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Leptin and Sirtinol were purchased from Sigma Aldrich (Saint Louis, MO). All cell culture reagents, with the exception of fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and antibiotic/antimycotic mix (Sigma Aldrich, Saint Louis, MO) were purchased from Invitrogen (Carlsbad, CA). Human SH-SY5Y neuroblastoma cells were purchased from ATCC (Manassas, VA).

Marwarha G., Raza S., Meiers C, & Ghribi O. (2014). Leptin attenuates BACE1 expression and Amyloid-β genesis via the activation of SIRT1 signaling pathway. Biochimica et biophysica acta, 1842(9), 1587-1595.

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Cultivation and Characterization of Colorectal Cancer Cell Lines

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HCT116, SW620, and HT29 CRC cell lines were a kind gift from Prof. Kari Alitalo, University of Helsinki, Finland. SW1222 cells and normal human colon fibroblasts (ATCC-1459) were from ECACC and ATCC, respectively. Thp-1 cells were from ATCC. Cells were cultured in DMEM high glucose (Gibco) supplemented with 10% FCS (Gibco), cyprofloxacine, antibiotic/antimycotic mix, and glutamine (Sigma). Cells were tested for mycoplasma contamination with Hoechst staining and only negative cultures were used in our experiments. Two days before EV collection, cells were washed with PBS three times and they were further cultured in either medium without FBS or containing 2.5% EV-free FBS. EV-free FBS was prepared by overnight ultracentrifugation at 100,000g or purchased from Gibco (exosome depleted One-Shot FBS). For 3D cultures, cells were treated with TrypLE (Gibco), embedded into Matrigel with 5000–10,000 cells depending on the cell line and cultured for 12-14 days.

Szvicsek Z., Oszvald Á., Szabó L., Sándor G.O., Kelemen A., Soós A.Á., Pálóczi K., Harsányi L., Tölgyes T., Dede K., Bursics A., Buzás E.I., Zeöld A, & Wiener Z. (2019). Extracellular vesicle release from intestinal organoids is modulated by Apc mutation and other colorectal cancer progression factors. Cellular and Molecular Life Sciences, 76(12), 2463-2476.

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Porcine Vascular Tissue Culture

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Abattoir pig vessels largely from white X female pigs aged 8–10 weeks old were obtained from the Royal Veterinary College. Fresh tissue was collected and used within 4 h. The 50–500 mg segments of vessel or perivascular aortic adventitia were cultured in serum-free DMEM supplemented with antibiotic/antimycotic mix (Sigma-Aldrich, St. Louis, MO., USA) at 37 °C, 5% CO2 and 95% air, as previously described for rat and human vessels [56 (link),57 (link)]. Serum-free media was used, as most sera contain large amounts of oxylipins (unpublished observations). Organ culture was performed for just the first 24 h after explant in order to minimize cell differentiation. In some experiments, lipopolysaccharide (LPS; E. coli, 1 μg/mL) was given to induce an inflammatory response.

Edin M.L., Lih F.B., Hammock B.D., Thomson S., Zeldin D.C, & Bishop-Bailey D. (2020). Vascular Lipidomic Profiling of Potential Endogenous Fatty Acid PPAR Ligands Reveals the Coronary Artery as Major Producer of CYP450-Derived Epoxy Fatty Acids. Cells, 9(5), 1096.

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Human Coxsackievirus A10 Cultivation

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The human rhabdomyosarcoma cell line (RD) was purchased from the ATCC. The cells were cultivated in DMEM (Sigma Aldrich) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% antibiotic–antimycotic mix (Sigma Aldrich) in a 37°C incubator (Sanyo; CO₂: 5%, relative humidity: >95%). A medium containing 1% antibiotic–antimycotic mix and 0% or 2% fetal bovine serum was used for infection and propagation, respectively. Human Coxsackievirus A10 was obtained from the ATCC and grown on RD cells. The stocks were frozen at −80°C and virus titers were determined by TCID₅₀ assay.

Morokutti-Kurz M., Graf C, & Prieschl-Grassauer E. (2017). Amylmetacresol/2,4-dichlorobenzyl alcohol, hexylresorcinol, or carrageenan lozenges as active treatments for sore throat. International Journal of General Medicine, 10, 53-60.

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Monocyte-derived dendritic cell differentiation

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Isolated monocytes were resuspended in 10% FBS RPMI medium containing 20 mM HEPES, 50 μM β-mercaptoethanol, 1× antibiotic antimycotic mix (Sigma, Moscow, Russia) and supplemented with 10 ng/ml of human GM-CSF and human IL-4 (both from ProSpec-Tany technoGene, Ness Ziona, Israel). Monocytes were seeded in 24 well plates at concentration of 2 × 10⁵ cell/well and stimulated in the absence (DMSO) or presence of 30 μM stable adenosine analog NECA for 3 days at 37°C in CO₂-incubator.

Yuryeva K., Saltykova I., Ogorodova L., Kirillova N., Kulikov E., Korotkaya E., Iakovleva Y., Feoktistov I., Sazonov A, & Ryzhov S. (2015). Expression of adenosine receptors in monocytes from patients with bronchial asthma. Biochemical and biophysical research communications, 464(4), 1314-1320.

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HeLa Cell Cultivation and Virus Propagation

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The human cervical epithelial carcinoma cell line (HeLa) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, Vienna, Austria) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% antibiotic–antimycotic mix (Sigma Aldrich) in a 37°C incubator (Sanyo, Okayama Prefecture, Japan; CO₂: 5%, relative humidity: >95%). In all experiments a medium containing 2% fetal bovine serum and 1% antibiotic–antimycotic mix was used. HRV1a and 8 serotypes were obtained from the ATCC and grown on HeLa cells. The stocks were frozen at −80°C and virus titers were determined by 50% tissue culture infective dose (TCID₅₀) assay.

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