Penicillin streptomycin neomycin antibiotic mixture

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Penicillin-streptomycin-neomycin antibiotic mixture is a solution containing three broad-spectrum antibiotics commonly used in cell culture applications. It is designed to prevent bacterial contamination in cell culture media and samples.

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26 protocols using penicillin streptomycin neomycin antibiotic mixture

Primary Human Hepatocyte Infection Assay

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Primary human hepatocyte infection in this study followed published methods (Roth et al., 2018 (link)). Briefly, cryopreserved primary human hepatocytes (BioIVT) were thawed two days prior to infection and seeded with 18,000 cells per well in a 384-well collagen-coated plate (Greiner). Hepatocyte cultures were maintained with daily media change with customized InVitroGro HI medium without dexamethasone (BioIVT) supplemented with 5% human serum (Interstate Blood Bank), 1:100 dilution of penicillin/streptomycin/neomycin antibiotic mixture (Gibco), and 1:1000 dilution of gentamicin (stock concentration:10 mg/mL, Gibco). The salivary glands of P. falciparum BD007 infected A. stephensi mosquitoes were collected between 18-20 days post-infection and were passed through a 30-gauge needle (Becton Dickinson). Total sporozoites were determined using a hemocytometer, and 24,000 sporozoites were inoculated into the hepatocyte culture. The plate was centrifugated at room temperature at 250 rcf for 5 min with acceleration/break at 5. Hepatocyte cultures were kept at 38°C with 5% CO₂ (Panasonic). These cultures were treated with human IFN_α and IFN_β (PBL Assay Science) at varying concentrations, 1U = 3.6pg.

Marques-da-Silva C., Peissig K., Walker M.P., Shiau J., Bowers C., Kyle D.E., Vijay R., Lindner S.E, & Kurup S.P. (2022). Direct type I interferon signaling in hepatocytes controls malaria. Cell reports, 40(3), 111098.

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Cryopreserved Primary Human Hepatocyte Infection

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Primary human hepatocyte infection in this study followed published methods [34 (link)]. Briefly, cryopreserved primary human hepatocytes (BioIVT, Westbury, NY, USA) were thawed two days prior to infection and seeded with 1.8 × 10³ cells per well in a 384-well collagen-coated plate (Greiner Bio-One). Hepatocyte culture was maintained with daily media change with customized InVitroGro HI medium without dexamethasone (BioIVT) supplemented with 5% human serum (Interstate Blood Bank, Memphis, TN, USA), 1:100 dilution of penicillin/streptomycin/neomycin antibiotic mixture (Gibco, Hampton, NH, USA), and 10 μg/mL gentamicin (Gibco). 1 × 10³ sporozoites were inoculated into the hepatocyte culture, and the plate was centrifuged at 250 g, at room temperature, and for 5 min. Hepatocyte cultures were kept at 38 °C with 5% CO₂ in an incubator (Panasonic, Kadoma, Osaka, Japan).

Bowers C., Hancox L., Peissig K., Shiau J.C., Vantaux A., Witkowski B., Phal S., Maher S.P., Harty J.T., Kyle D.E, & Kurup S.P. (2022). Cryopreservation of Plasmodium Sporozoites. Pathogens, 11(12), 1487.

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Primary Human Hepatocyte Infection Assay

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Silk Fibroin-Reinforced PLLA Scaffolds

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Poly-l-lactic acid (PLLA, M_w: 50 000, Daigang Biomaterial, Jinan,
China); methylene chloride (Fengchuan Chemical Reagent Co., Tianjin,
China); ammonium bicarbonate (Adamas-β, Shanghai, China); poly(vinyl
alcohol) (PVA, Sinopec Chongqing Svw Chemical Co., Chongqing, China);
NaOH (Fengchuan Chemical Reagent Co., Tianjin, China); and degummed
silk and SF (Beijing Sinolactide Medical Technology Company, China).
Gelatin (Macklin, Shanghai, China); carbodiimide (EDC) (Aladdin, Shanghai,
China); type IV collagenase (Sigma-Aldrich, MO); high-glucose Dulbecco’s
modified Eagle’s medium (H-DMEM, Gibco, CA); fetal bovine serum
(FBS, Gibco, CA); penicillin–streptomycin–neomycin antibiotic
mixture (PSN, Gibco, CA); LIVE/DEAD Viability/Cytotoxicity Kit (Thermo
Fisher, CA); CCK-8 solution (Beyotime, Shanghai, China); TRIzol reagent
(Thermo Fisher, CA); Go Taq qPCR and RT-qPCR systems (Promega); SYBR
Green Kit (Roche, Germany); 4% paraformaldehyde solution (Beyotime,
Shanghai, China); sucrose (Sigma, MO); optimum cutting temperature
(OCT) compound (Sakura, Japan); anti-collagen I antibody (Abcam, U.K.);
anti-collagen II antibody (Abcam, U.K.); secondary antibody (Solarbio,
Beijing, China); and DAB detection kit (Solarbio, Beijing, China).

Zeng Y., Li X., Liu X., Yang Y., Zhou Z., Fan J, & Jiang H. (2021). PLLA Porous Microsphere-Reinforced Silk-Based Scaffolds for Auricular Cartilage Regeneration. ACS Omega, 6(4), 3372-3383.

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Influenza Virus Replication Optimization

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The IFN-competent human cells include alveolar epithelial cell line A549 and were obtained from central public health laboratories in Egypt (CPHL), Madin-Darby canine kidney cells (MDCK II), European Collection of Cell Cultures (ECACC), London, UK, recommended by World Health Organization for influenza virus replication and were obtained from central public health laboratories in Egypt (CPHL), and were kept in maintenance medium containing Dulbecco's Modified Eagle Medium, (DMEM; Gibco, Paisley, UK), 2 mM l-Glutamine (Gibco), 1× NEAA (Nonessential Amino Acids, Gibco), and 24 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, Gibco) at 30°C. MDCK II cells were subcultured at 3-4 × 10⁴ cells/cm² with growth medium maintenance medium supplemented with 10% fetal bovine serum (FBS; Gibco). Growth medium was supplemented with 1 mg/mL Geneticin® (Gibco) for cell line continuation or with 2.5 µg/mL Fungizone® (Gibco) and 1× PSN (Penicillin-Streptomycin-Neomycin Antibiotic Mixture, Gibco) when cells were plated to be infected. Cultures were incubated at 37°C for 48 h prior to infection to reach a minimum of 90% monolayer confluence.

El-Sayed I., Bassiouny K., Nokaly A., Abdelghani A.S, & Roshdy W. (2016). Influenza A Virus and Influenza B Virus Can Induce Apoptosis via Intrinsic or Extrinsic Pathways and Also via NF-κB in a Time and Dose Dependent Manner. Biochemistry Research International, 2016, 1738237.

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Collagen Gel Elasticity Analysis of HDFs

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HDFs derived from infant skin were isolated as described previously (Aiba-Kojima et al. 2007 ) and were cultured with Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries) containing 10% fetal bovine serum (Sigma-Aldrich) and a penicillin-streptomycin-neomycin antibiotic mixture (1×; Gibco). HDFs were used at passage numbers 5-9. For analysis of collagen gel elasticity, type I collagen was prepared in-house from rat tails based on a previously reported method (Yamato et al. 1992 ) and type III collagen from bovine skin was purchased from Nippi (PSC-3-100-100; Tokyo, Japan). The 2D collagen gel (0.3%) was prepared by mixing type I and III collagen solutions (5.0 mg/mL), 10× medium and reconstruction buffer (0.02 M HEPES, 0.03 M NaHCO 3 , 0.05 M NaOH). The ratios of type III collagen to total collagen were 0%, 10%, 20%, 40%, 60% and 80%. HDFs were cultured on 2D collagen gels.

Ohto-Fujita E., Shimizu M., Sano S., Kurimoto M., Yamazawa K., Atomi T., Sakurai T., Murakami Y., Takami T., Murakami T., Yoshimura K., Hasebe Y, & Atomi Y. (2019). Solubilized eggshell membrane supplies a type III collagen-rich elastic dermal papilla. Cell and tissue research, 376(1).

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Procyanidin A2 Effects on Alveolar Cells

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The human alveolar epithelial (A549) cell-line was purchased from the American Type Culture Collection (ATCC^® CCL-185™, (c/o Cryosite, New South Wales, Australia)). Cell culture media, phosphate buffered saline (PBS), penicillin-streptomycin-neomycin antibiotic mixture, 100× l-glutamine, and 2.5% trypsin were purchased from Life Technologies (Auckland, New Zealand). Fetal bovine serum (FBS) was purchased from Moregate Biotech (Hamilton, New Zealand). Procyanidin A2 (HPLC ≥ 99%, epicatechin-(4β-8, 2β-O-7)-epicatechin) was purchased from Extrasynthese (Genay Cedex, France). The CCL26 DuoSet ELISA kit, human recombinant IL-4 and IFNγ were purchased from R&D Systems (Pharmaco, Auckland, New Zealand). Low-endotoxin bovine serum albumin was purchased from MP Biomedicals (Auckland, New Zealand). Ecoteric T20 (Tween 20) and 30% hydrogen peroxide were purchased from ThermoFisher Scientific (Auckland, New Zealand). Sodium acetate tri-hydrate was from BDH Laboratory Supplies (Poole, UK). β-nicotinamide adenine dinucleotide reduced dipotassium salt (NADH), dimethyl sulfoxide (DMSO) and all other chemicals not specifically listed were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Coleman S.L., Kruger M.C., Sawyer G.M, & Hurst R.D. (2016). Procyanidin A2 Modulates IL-4-Induced CCL26 Production in Human Alveolar Epithelial Cells. International Journal of Molecular Sciences, 17(11), 1888.

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Isolation and Expansion of Rat Bone Marrow Stromal Cells

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Young adult (4 weeks old) Sprague–Dawley rats were anesthetized with isoflurane (2–3% in O₂). After decapitation, both femurs were removed and placed in a dish containing sterile Hank’s buffered salt solution (HBSS) on ice. The ends of the femurs were removed and bone marrow extruded into sterile HBSS using an 18-G cannula attached to a 12-ml syringe. Extruded bone marrow was dispersed by trituration and centrifuged at 400g for 10 min, repeated twice. The cells were then resuspended in culture medium consisting of low-glucose Dulbecco’s Modified Eagle Medium supplemented with 10% stem cell-qualified fetal bovine serum and penicillin–streptomycin–neomycin antibiotic mixture (Life Technologies, Carlsbad, CA) and placed in culture. The flasks were washed twice at a 24-h interval to eliminate non-adherent cells. Adherent cells were further cultured in 25-cm² flasks with medium change at 2–3 day intervals. Prior to cell transplantation, the cultured BMSCs 10–12 days (without any passage after harvest) after isolation were digested using TrypLE (Life Technologies) and centrifuged at 400g for 5 min. The pelleted BMSCs were resuspended in sterile phosphate-buffered saline (PBS). Cell viability was determined by trypan blue exclusion and cell numbers counted by hemocytometer.

Fischer G., Wang F., Xiang H., Bai X., Yu H, & Hogan Q.H. (2017). Inhibition of neuropathic hyperalgesia by intrathecal bone marrow stromal cells is associated with alteration of multiple soluble factors in cerebrospinal fluid. Experimental brain research, 235(9), 2627-2638.

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Culturing Primary Melanocytes and Melanoma Cells

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The human primary neonatal foreskin melanocyte cell line HEMn-LP from a lightly pigmented donor was cultured at 37 °C and 5% CO2 in Medium 254 supplemented with human melanocyte growth supplement (HMGS). Melanocyte cell line, medium, and supplement listed above were purchased from Invitrogen (Carlsbad, CA, USA). The human melanoma cell line MDA-MB 435 was purchased from the American Tissue Cell Culture Collection (ATCC, Manassas, VA, USA), and the melanoma cell lines A2058 and A375 were obtained from Dr. Shing-Chuan Shen (Taipei Medical University, Taipei, Taiwan). All melanoma cell lines were maintained at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin-neomycin (PSN) antibiotic mixture (Gibco).

Nguyen H.D., Liao Y.C., Ho Y.S., Chen L.C., Chang H.W., Cheng T.C., Liu D., Lee W.R., Shen S.C., Wu C.H, & Tu S.H. (2019). The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration. Cancers, 11(12), 1991.

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Dissection and Culture of Ae. aegypti Midgut Cells

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Mosquitoes were cold anaesthetized, and all dissections were performed under sterile conditions following previously described protocols [19 (link),20 (link)] with minor modifications. In brief, the mosquitoes were surface sterilized with a 70% ethanol solution [21 (link)] and rinsed with sterile phosphate-buffered saline (PBS). Ae. aegypti midguts were carefully dissected [22 ] under a dissecting microscope (15× magnification). The midguts were immersed in sterile PBS solution supplemented with 1× penicillin‑streptomycin-neomycin (PSN) antibiotic mixture (Gibco^®, Life Technologies) and trypsinized with 1× Krebs-Ringer bicarbonate solution supplemented with 0.25% trypsin (37 °C for 25 minutes). The cells were pelleted (500× g, 4 °C, 15 min), resuspended in MEM media supplemented with 10% FBS and incubated in 96-well plates (28 °C, 5% CO₂) with one passage every 12 days.

Tham H.W., Balasubramaniam V.R., Tejo B.A., Ahmad H, & Hassan S.S. (2014). CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells. Viruses, 6(12), 5028-5046.

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