Penicillin g

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Penicillin G is a broad-spectrum antibiotic used in various laboratory and research applications. It is a naturally occurring compound produced by the Penicillium fungus. Penicillin G is effective against a wide range of Gram-positive and some Gram-negative bacteria.

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883 protocols using penicillin g

Comprehensive Cell Line Culture Protocols

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Human cancer cell lines HL-60, K562, Jurkat, HeLa, A549, MCF-7, HepG2, A431, and MIA PaCa-2, along with human normal cell lines WI-38 and 1C3D3, were obtained from the RIKEN Cell Bank (RIKEN BRC, Tsukuba, Japan). Human normal cell line YS-1 was obtained from the JCRB Cell Bank (Osaka, Japan). Human cancer cell lines U937, PC-3, DLD-1, Hep3B, WM266-4, SK-MEL-28, HT-1080, and BxPC-3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HL-60, K562, Jurkat, U937, MCF-7, DLD-1, and BxPC-3 cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (Sigma-Aldrich), 50 units/mL penicillin G (Gibco), and 50 μg/mL streptomycin (Gibco). HeLa, A549, PC-3, HepG2, Hep3B, WM266-4, SK-MEL-28, HT-1080, A431, MIA PaCa-2, and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal calf serum, 50 units/mL penicillin G, and 50 μg/mL streptomycin. YS-1 cells were cultured in DMEM containing 5% fetal calf serum, 50 units/mL penicillin G, and 50 μg/mL streptomycin. 1C3D3 cells were cultured in DMEM containing 5% fetal calf serum, 10% newborn bovine serum (SAFC Biosciences, Lenexa, KS, USA), 2.5% horse serum (Gibco), 50 units/mL penicillin G, and 50 μg/mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

KAWATANI M., AONO H., HIRANUMA S., SHIMIZU T., MUROI M., NOGAWA T., OHISHI T., OHBA S.I., KAWADA M., YAMAZAKI K., DAN S., DOHMAE N, & OSADA H. (2023). Identification of a dihydroorotate dehydrogenase inhibitor that inhibits cancer cell growth by proteomic profiling. Oncology Research, 31(6), 833-844.

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T Cell Stimulation and Culture Protocols

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Human T cells were stimulated with plate-bound anti-CD3 (1 µg/ml) and soluble anti-CD28 (1 µg/ml) (BD Bioscience) antibodies in RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM l-glutamine (all from Life Technologies Invitrogen), 10% normal human serum (NHS), and 10 U/ml IL-2 (Novartis) for the indicated amount of time.
Murine T cells were cultured without stimulation or stimulated with plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (10 µg/ml) (BD Bioscience) antibodies in RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM l-glutamine, 10% FCS (all from Life Technologies Invitrogen), and 10 ng/ml IL-2 (R&D Systems) for the indicated amount of time. Mouse TGFβ1 (2 ng/ml, R&D Systems) was added when indicated. For serum starvation, cells were incubated with serum-free RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM l-glutamine (Life Technologies Invitrogen) and 10 ng/ml IL-2 (R&D Systems) for 20 h.

Lehmkuhl P., Gentz M., Garcia de Otezya A.C., Grimbacher B., Schulze-Koops H, & Skapenko A. (2021). Dysregulated immunity in PID patients with low GARP expression on Tregs due to mutations in LRRC32. Cellular and Molecular Immunology, 18(7), 1677-1691.

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Propagation of Influenza A(H1N1) in MDCK Cells

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Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) and influenza A(H1N1) strain A/WS/33 (ATCC VR-825) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Complete growth medium for MDCK cells consisted of Eagle’s Minimum Essential Medium (EMEM) (ATCC) containing 10% fetal bovine serum (Hyclone Laboratories Inc, Logan, UT), 200 units/ml penicillin G, 200 μg/ml streptomycin (Invitrogen, Carlsbad, CA). MDCK cells were incubated at 35 °C in a humidified 5% CO₂ incubator until approximately 80% confluent. Propagation of influenza A(H1N1) [1.0×10⁷ TCID₅₀] and dilution in Viral Transport Media (VTM) consisting of Hank’s Balanced Salt Solution (1X HBSS; ThermoFisher Scientific) supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), 100 units/ml penicillin G and 100 units/ml streptomycin (ThermoFisher Scientific), was performed as previously described (Blachere et al., 2011 (link)).

Blachere F.M., Lindsley W.G., McMillen C.M., Beezhold D.H., Fisher E.M., Shaffer R.E, & Noti J.D. (2018). Assessment of influenza virus exposure and recovery from contaminated surgical masks and N95 respirators. Journal of virological methods, 260, 98-106.

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Cell Culture Protocol for Human Medulloblastoma and HEK293T

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D341 (ATCC^® HTB-187TM, RRID:CVCL_0018) and HEK293T (ATCC^® CRL-3216, RRID:CVCL_0063) were purchased from ATCC, and D425 (SCC290, RRID:CVCL_1275) was purchased from Sigma-Millipore (Sigma-Aldrich, St. Louis, MO, USA) [54 (link),55 (link),56 (link)]. Human medulloblastoma cells were grown in Minimum Essential Media (MEM; Gibco 11095; Thermo Fisher Scientific, Waltham, MA, USA) plus 15% fetal bovine serum (FBS; Gibco 26140079; Thermo Fisher Scientific, Waltham, MA, USA), 100 mg/mL penicillin G, and 100 lg/mL streptomycin (Gibco 15140122). HEK293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco 11965092) supplemented with 10% FBS (Gibco 26140079), 100 mg/mL penicillin G, and 100 lg/mL streptomycin (Gibco 15140122; Thermo Fisher Scientific, Waltham, MA, USA). All cell lines were cultured at 37 °C in a humidified environment with 5% CO₂ and confirmed mycoplasma negative (Lonza MycoAlert LT07-318; Basel, Switzerland) (most recently confirmed negative in January 2024).

Ampudia-Mesias E., Cameron C.S., Yoo E., Kelly M., Anderson S.M., Manning R., Abrahante Lloréns J.E., Moertel C.L., Yim H., Odde D.J., Saydam N, & Saydam O. (2024). The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas. International Journal of Molecular Sciences, 25(8), 4416.

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Colorectal Cancer Cell Line Maintenance

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Human colorectal cancer cell lines DLD‐1 and SW620 were used for all assays (American Type Culture Collection [ATCC]). DLD‐1 cells were maintained in McCoy's 5A modified medium (ThermoFisher Scientific) supplemented with 10% foetal bovine serum (FBS, Gibco, ThermoFisher Scientific), 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate (ThermoFisher Scientific). SW620 cells were maintained in Dulbecco's modified Eagle medium (DMEM, Sigma‐Aldrich) supplemented with 10% FBS (Gibco, ThermoFisher Scientific), 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate (ThermoFisher Scientific) and 2 mM GlutaMAX (ThermoFisher Scientific). Cells were cultured at 37°C and 5% CO₂/air.

Rees P.A., Castle J., Clouston H.W., Lamb R., Singh U., Duff S.E, & Kirwan C.C. (2023). The effects of coagulation factors and their inhibitors on proliferation and migration in colorectal cancer. Cancer Medicine, 12(16), 17184-17192.

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Culturing Rat PC12 and Mouse Stem Cells

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Rat pheochromocytoma PC12 cells were kindly provided by Dr. Irene Yan (Department of Histology, University of São Paulo, SP, Brazil). The culture was maintained essentially as previously described [23] (link), in Dulbecco's modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific), 5% horse serum (HS) (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific), 100 mg/mL streptomycin sulphate, and 100 U/mL penicillin G (Thermo Fisher Scientific). Mouse embryonic stem (ES) USP-4 cells were maintained over a murine embryonic fibroblast (MEF) feeder layer [24] (link). USP-4 and murine embryo-derived teratocarcinoma P19 cells [25] (link) were cultured with Minimum Essential Medium Eagle, -modification, with nucleosides (-MEM) (Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM l-glutamine, 100 mg/mL streptomycin sulphate, and 100 U/mL penicillin G, and maintained at 37 °C, in a 5% CO 2 atmosphere.

Trombetta-Lima M., Assis-Ribas T., Cintra R.C., Campeiro J.D., Guerreiro J.R., Winnischofer S.M.B., Nascimento I.C.C., Ulrich H., Hayashi M.A.F, & Sogayar M.C. (2021). Impact of Reck expression and promoter activity in neuronal in vitro differentiation. Molecular biology reports, 48(2).

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Culturing Human MSCs and OVCAR-3 Cells

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Human MSCs were purchased from the Tulane Centre for Gene Therapy, New Orleans, LA, USA, and were cultured in alpha minimum essential media (α-MEM) supplemented with 16% heat-inactivated fetal bovine serum (FBS), L-glutamine (4 mM), penicillin G (50 U/mL), and streptomycin (50 µg/mL), all of which were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Human OVCAR-3 ovarian carcinoma cancer cells were obtained from the Metabolic and Molecular Imaging Group, MRC Clinical Sciences Centre, Imperial College, London (originally from the ATCC^® HTB-161™). Cells were cultured in RPMI 1640 media (Thermo Fisher Scientific) supplemented with 10% FBS, L-glutamine (4 mM), penicillin G (50 U/mL), and streptomycin (50 µg/mL).
All cells were grown in T175 flasks (Thermo Fisher Scientific) in a humidified incubator at 37°C with 95% air and 5% CO₂. The cell lines used within this study have been purchased from known cell providers, and are an expansion of the original primary cell culture. These cell lines are therefore not considered relevant material under the Human Tissue Act and do not require ethics approval.

Kalber T.L., Ordidge K.L., Southern P., Loebinger M.R., Kyrtatos P.G., Pankhurst Q.A., Lythgoe M.F, & Janes S.M. (2016). Hyperthermia treatment of tumors by mesenchymal stem cell-delivered superparamagnetic iron oxide nanoparticles. International Journal of Nanomedicine, 11, 1973-1983.

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Culturing Human Cell Lines

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ASC52telo (hTERT immortalized human adipose-derived mesenchymal stem cells), MCF7 (human breast adenocarcinoma), MDA-MB-231 (human triple negative breast cancer), and MCF10A (human non-tumorigenic breast epithelial cells) cell lines were purchased from American Type Tissue Culture Collection (ATCC, Rockville, MD, USA). All cells were maintained at 37 °C under a humidified atmosphere containing 5% CO₂. ASC52telo cells were grown in Mesenchymal Stem Cell Basal Medium (ATCC) supplemented with 2% Fetal Bovine serum (FBS), 5 ng/mL rh FGF basic, 5 ng/mL rh FGF acidic, 5 ng/mL rh EGF, 2.4 mM L-Alanyl-L-Glutamine, 0.2 mg/mL G418, 10,000 U/mL penicillin G, 10 mg/mL streptomycin, and 25 ug/mL amphotericin B. MDA-MB-231 and MCF7 cells were grown using Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax^TM supplemented with 10% FBS, 100 U/mL streptomycin and 100 µg/mL penicillin G (Life Technologies, Carlsbad, CA, USA). MCF10A were grown using DMEM/F12 supplemented with 5% horse serum (Life Technologies), 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 µg/mL insulin (Sigma Aldrich, Milano, Italy).

Avancini G., Menilli L., Visentin A., Milani C., Mastrotto F, & Moret F. (2023). Mesenchymal Stem Cell Membrane-Coated TPCS2a-Loaded Nanoparticles for Breast Cancer Photodynamic Therapy. Pharmaceutics, 15(6), 1654.

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Culturing and Maintaining Cell Lines

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We purchased the following cell lines from American Type Culture Collection (ATCC): HCT116 (CCL-247), U2OS (HTB-96), HEK293T (CRL-3216), and RPE1 (CRL-4000). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), which contained 10% fetal bovine serum (FBS) (General Electric ≥ Healthcare), 100 U/mL penicillin G (Life Technologies), and 100 μg/mL streptomycin (Life Technologies). The patient-driven glioblastoma cell line GBL-67 and neural stem cell NSC-10 were established and maintained (35 (link)) and used in this study following the approval of the Institutional Review Board (IRB) at the Seoul National University Hospital (IRB No. H-1904-117-1028). These cells were cultured in DMEM, which contained 20% FBS (GE Healthcare), 100 U/mL penicillin G (Life Technologies), and 100 μg/mL streptomycin (Life Technologies) in a 5% hypoxia chamber.

Kwon T., Ra J.S., Lee S., Baek I.J., Khim K.W., Lee E.A., Song E.K., Otarbayev D., Jung W., Park Y.H., Wie M., Bae J., Cheng H., Park J.H., Kim N., Seo Y., Yun S., Kim H.E., Moon H.E., Paek S.H., Park T.J., Park Y.U., Rhee H., Choi J.H., Cho S.W, & Myung K. (2022). Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9. Proceedings of the National Academy of Sciences of the United States of America, 119(9), e2103532119.

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Cell Culture and Synchronization Protocols

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HeLa, U2OS, HeLa-ATAD5^{mNeonGreen-AID}, HeLa-ATAD5^AID, U2OS-ATAD5^AID, U2OS-ATAD5^−/− and control wild-type cells, HEK293AD, and HEK293AD-ATAD5^−/− cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS; GE Healthcare, Little Chalfont, UK), 100 U/ml penicillin G (Life Technologies, Carlsbad, CA, USA) and 100 μg/ml streptomycin (Life Technologies) in a humidified atmosphere of 5% CO₂ at 37°C. MRC-5 cells were cultured in Minimum Essential Medium with Earle’s Balanced Salts containing 10% FBS (GE Healthcare), 100 U/ml penicillin G (Life Technologies) and 100 μg/ml streptomycin (Life Technologies) in a humidified atmosphere of 5% CO₂ at 37°C. To generate HEK293AD-ATAD5^−/−: CLIP vector and HEK293AD-ATAD5^−/−: CLIP-ATAD5 cells, HEK293AD-ATAD5^−/− cells were infected by lentivirus generated using pLVX-CMV-CLIP or pLVX-CMV-CLIP-ATAD5 DNA and selected with puromycin. To enrich cells at the G1 phase, cells were treated with 1 μM CDK 4/6 inhibitor PD 0332991 for 24 h and 20 μM CDK 7/9 inhibitor PHA-767491 for 1 h before drug treatment. To obtain mitotic cells, cells were treated with 50 ng/ml nocodazole for 5 h, and then cells were shaken off the plate, collected and washed with culture medium. Cells were then incubated in fresh medium for 4 h (G1) before drug treatment.

Park S.H., Kim Y., Ra J.S., Wie M.W., Kang M.S., Kang S., Myung K, & Lee K.Y. (2021). Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair. Nucleic Acids Research, 49(20), 11746-11764.

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