Penicillin

Name: Penicillin
Brand: Thermo Fisher Scientific
SKU: SSLhCZIBPBHhf-iFeNIp
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50 197 citations

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About the product

Penicillin is a type of antibiotic used in laboratory settings. It is a broad-spectrum antimicrobial agent effective against a variety of bacteria. Penicillin functions by disrupting the bacterial cell wall, leading to cell death.

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The Thermo Scientific Chemicals brand offers various Penicillin products that are actively commercialized, including Penicillin G potassium salt, Penicillin G sodium salt, and Penicillin V potassium salt. These products are available in different quantities and concentrations suitable for research applications.

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50 197 protocols using «penicillin»

Isolation and Culture of hDPSCs

2025

Human third molars from dental surgery waste of young healthy donors between 18 and 30 years of age were selected from a bigger cohort of between 18- and 45-years old subjects, to reduce potential sample variability. Pulp extraction and hDPSCs primary culture were performed following previously reported protocols [13 (link), 22 (link)]. Briefly, after tooth fracture, the dental pulp was collected and digested with an enzymatic solution of 3 mg/mL collagenase (#17018029, Gibco) and 4 mg/mL dispase (#D4693-1G, Merck) for 1 h at 37°C. After centrifugation, each donor’s cell pellet was resuspended and cultured in parallel following two different culture media. On the one hand, we generated standard plastic adherent cultures on conventional tissue culture-treated flasks (#83.3912.002, Sarstedt) with DMEM (#D5796, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; #SV30160.03, HyClone), 100 U/mL penicillin and 150 mg/mL streptomycin (#11528876, Gibco) to generate adherent cell monolayers. On the other hand, a serum-free culture medium was combined with low binding adhesion surfaces (#3814, Corning), using Neurocult basal media supplemented with human Neurocult proliferation supplement (#05751, Stem Cell Technologies), both at 9:1 ratio, and supplemented with Heparin solution 2 µg/mL (#07980, Stem Cell Technologies), EGF 20 ng/mL, and bFGF 10 ng/mL (Peprotech, London, United Kingdom), 2% B-27 without vitamin A (#12587010, Thermo Fisher), 2 mM GlutaMAX (#11500626, Fischer Scientific), 100 U/mL penicillin and 150 mg/mL streptomycin (#11528876, Gibco) to generate free-floating neurogenic dentospheres.

Pardo-Rodríguez B., Baraibar A.M., Manero-Roig I., Luzuriaga J., Salvador-Moya J., Polo Y., Basanta-Torres R., Unda F., Mato S., Ibarretxe G, & Pineda J.R. (2025). Functional differentiation of human dental pulp stem cells into neuron-like cells exhibiting electrophysiological activity. Stem Cell Research & Therapy, 16, 10.

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Quantitative Proteomic Profiling of Zika Virus Infection

2025

For the comparison of ZIKV-infected to Mock-infected immortalized fibroblasts, cells were cultured and infected at the Department of Clinical Microbiology (Umeå) as described above (Transcriptomics experiments). For the comparison of two primary cell lines (F02-09, F10-09), a kind gift from Prof. Thomas Werfel, to the immortalized cell line (hTert), all cells were cultured at the Research Center for Emerging Infections and Zoonoses (Hannover) in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS Advanced, Capricorn Scientific), 1% non-essential amino acids, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all Thermo Fisher). A commercially available HeLa- (Thermo Fisher) and a self-made HEK-cell-derived protein digest standard were used as references. Of each condition/cell line, 4 technical replicates were provided for sample processing and analysis.
Cells were lysed with RIPA buffer on ice for 30 min. Cellular debris were removed by centrifugation for 10 min at 12,000 g and 4 °C and transferring the supernatant to a new reaction tube. 20 µg of protein were taken from the lysate for precipitation overnight using ice cold acetone (final concentration of 80%). Proteins were spun down at 11,000 g for 15 min at 4 °C. Digest of proteins was performed using Trypsin Gold (Promega) following the manufacturer’s instructions. Samples were purified using StageTips following the protocol of Rappsilber et al.^{39 (link)}. Purified peptides were dried in a SpeedVac Vacuum Concentrator and resuspended in MS buffer (0.1% formic acid, 5% acetonitrile in water (MS grade)). Samples were analysed via liquid chromatography–mass spectrometry (LC-MS) using a Vanquish Neo nanoflow UHPLC System coupled to an Orbitrap Eclipse mass spectrometer (Thermo Scientific™). Of each sample, 500 ng peptides were separated on a reversed-phase nanoViper™ PepMap™ separating column (150 mm length, 75 μm inner diameter, and 2 μm C18 particle size (Thermo Scientific™)) using buffer A (80% ACN, 0.1% FA) and B (80% ACN, 0.1% FA). Peptides were eluted at a flow rate of 250 nL/min at 40 °C by a gradient starting with Buffer B increased from 6% to 25% within 60 min and further increased to 90% within 10 min and held at 90% for an additional 8 min. Ionisation was achieved by a Nano Spray Flex Ion Source and stainless steel emitters (40 mm, OD 1/32) at 1,900 V. Precursor scans were performed in the Orbitrap mass analyser with an m/z range set to 375–1500, at a resolution of 120,000, and stored in profile mode. The twenty most intense precursors with intensities above 2000 counts were fragmented in the linear ion trap by higher-energy collisional dissociation with collision energy set to 30% and dynamic exclusion set to 45 s. Fragment ion scans were performed in the Ion Trap mass analyser with AGC set to Standard, Injection Time Mode set to Auto, mass range set to Normal and spectra stored in centroid mode.
MS raw data were processed with MaxQuant software (V. 2·4·14·0) for the identification and quantification of proteins using preconfigured settings and, additionally, selecting label-free quantification via LFQ (Min. ratio count set to 1) and IBAQ as well as writing mzTab tables. MS spectra were searched against the UniProt database of human (ID UP000005640, reviewed, database downloaded on 04/03/2024) and ZIKV proteins (Uniprot entry A0A1Z2X283).
The results were analysed with the Perseus software (V.1·6·15·0, Max Planck Institute of Biochemistry^{37 (link)}) based on LFQ values and applying preconfigured settings. From the resulting protein list, contaminants, proteins only identified by site modifications, proteins identified in the decoy database and proteins identified by only a single peptide were removed as well as bad replicates with less than 1000 quantified proteins. Further analyses were restricted to proteins with at least 2 valid values per group. Missing values were replaced by low numbers according to the normal distribution of the dataset. Groups were compared by pairwise t-tests. For Hierarchical Clustering analysis, intensities were further transformed to z-Scores. Enrichment analysis was performed using the web-based tool Metascape^{40 (link)}.

Mozūraitis R., Cirksena K., Raftari M., Hajkazemian M., Mustapha Abiodun M., Brahimi J., Radžiutė S., Apšegaitė V., Bernotienė R., Ignatowicz L., Hick T., Kirschning A., Lenman A., Gerold G, & Emami S.N. (2025). Zika virus modulates human fibroblasts to enhance transmission success in a controlled lab-setting. Communications Biology, 8, 139.

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Cell Line Characterization and Manipulation

2025

The human endometrial endothelial cell line, HEEC, and the human ESCC cell lines, EC109, KYSE-150, KYSE-510 and TE-3, were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The TE-3 cell line has been shown to be identical to the following cell lines: TE-2, TE-7, TE-12 and TE-13 (https://www.cellosaurus.org/CVCL_9971) (28 (link),29 (link)). The EC109 cells used in the present study were authenticated using short tandem repeat analysis (Table SI). Cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (AccuRef Scientific), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultured under an atmosphere of 5% CO₂ at 37°C. The concentration of nucleic acid was 20 nM and transfection was performed at room temperature, while subsequent experiments were conducted 48 h after transfection.
The KYSE-150 cells were transfected with an AGTR1-expressing pcDNA3.1 plasmid or empty vector (Thermo Fisher Scientific, Inc.) or with AGTR1-targeting small interfering (si)RNA oligonucleotides (antisense strand, 5′-CUG UAG AAU UGC AGA UAU UdT dT-3′ and sense strand, 3′-dTd TGA CAU CUU AAC GUC UAU AA-5′) or negative control (NC) oligonucleotides (antisense strand, 5′-UUC UCC GAA CGU GUC ACG UdT dT-3′ and sense strand, 3′-dTd TAA GAG GCU UGC ACA GUG CA-5′) (Shanghai GenePharma Co., Ltd.). Cell transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. After 48 h of transfection, cells were harvested for further analysis.
The Tet-on cell line with inducible expression of AGTR1 was constructed by transfection of KYSE-150 cells with the AGTR1-expressing pCDH plasmid (Vazyme Biotech Co., Ltd.) using Lipofectamine 3000 reagent as aforementioned and subsequent screening by limiting dilution cloning. AGTR1-expression was induced by treating the cells with 100 ng/ml doxycycline (MilliporeSigma). In some experiments, cells were treated with cisplatin (MilliporeSigma) at the indicated concentration and/or 5 µM fendiline (Thermo Scientific, Inc.) as specified.

Liu K., Bie J., Zhang R., Xiong R., Peng L., Luo Y., Yang S., Feng G, & Song G. (2025). AGTR1 potentiates the chemotherapeutic efficacy of cisplatin in esophageal carcinoma through elevation of intracellular Ca2+ and induction of apoptosis. International Journal of Oncology, 66(4), 32.

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Isolation and Culture of Diverse Cell Types

2025

Cell lines including African green monkey kidney (Vero), human hepatocellular carcinoma (HepG2), murine melanoma (B16), colorectal carcinoma (CT26), and mammary carcinoma (4T1) were sourced from the Peking Union Medical College Hospital. These cell types were maintained in Dulbecco’s Modified Eagle Medium (DMEM) from Gibco, enriched with 10% fetal bovine serum (FBS) supplied by Gibco, along with 100 IU/mL of penicillin and 100 µg/mL of streptomycin, both also from Gibco. The cells were incubated at 37 °C under a humidified environment with a 5% CO₂ atmosphere.
Bone marrow-derived macrophages (BMDMs) were generated from female C57BL/6 mice, aged 4–6 weeks. First, bone marrow was extracted by flushing the tibias and fibulas of the mice with phosphate-buffered saline (PBS), and then red blood cells were lysed using ammonium-chloride-potassium lysing buffer (ACK lysis buffer). Subsequently, the remaining cells were pelleted by centrifugation at 300 × g for 5 min. These cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 25 ng/mL murine macrophage colony-stimulating factor (M-CSF) for 6 days.
Bone marrow-derived dendritic cells (BMDCs) were produced using females of the C57BL/6 strain, aged 4–6 weeks, according to the following protocol. Initially, bone marrow was harvested by flushing the tibias and fibulas of the mice with PBS, followed by the lysis of red blood cells using ammonium-chloride-potassium lysing buffer (ACK lysis buffer). Afterward, the residual cells were pelleted by centrifugation at 300 × g for a duration of 5 min. These cells were then seeded at a density of 5 × 10⁵ cells per well in 24-well plates and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 20 ng/mL of murine interleukin-4 (IL-4) supplied by Peprotech, and 20 ng/mL of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) also from Peprotech, for a period of 7 days.
T lymphocytes were derived from the spleens of 4-6-week-old female C57BL/6 mice. The spleens were excised and mechanically disrupted to create a single-cell suspension. This suspension was then passed through 75 μm filters to remove any clumps. Following two washing steps, the red blood cells were eliminated by incubation with ACK lysis buffer. The remaining cells were sedimented by centrifugation at 800 × g for a period of 5 min. The purified cells were subsequently cultured at a density of 5 × 10⁵ cells per well in 24-well plates, using RPMI 1640 medium as the culture medium.

Wang Z., Chen H., Ming R., Wang W., Liu S., Jing Y., Yan Z., Lu G, & Huang L.L. (2025). A self-adjuvant multiantigenic nanovaccines simultaneously activate the antiviral and antitumor immunity for the treatment of cancers. Journal of Nanobiotechnology, 23, 150.

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Culturing HEK293T and 3T3-L1 Cells

2025

HEK293T (ATCC #CRL-11268) and 3T3-1L (ATCC #CL-173) cells were cultured according to ATCC guidelines. HEK293T cells were grown at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle Medium, high glucose, GlutaMAX Supplement (DMEM + GlutaMAX, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 units/mL of penicillin, 100 μg/mL of streptomycin (Gibco) until 90–95% confluence in T-75 flasks prior to subculture or use. 3T3-L1 cells were grown at 37 °C and 5% CO₂ in DMEM, high glucose, GlutaMAX, pyruvate (Gibco) supplemented with 10% bovine calf serum (BCS, Gibco) and 100 units/mL of penicillin, 100 μg/mL of streptomycin (Gibco) until 70% confluence in T-75 flasks prior to subculture or use.

MacTavish B.S., Zhu D., Shang J., Shao Q., He Y., Yang Z.J., Kamenecka T.M, & Kojetin D.J. (2025). Ligand efficacy shifts a nuclear receptor conformational ensemble between transcriptionally active and repressive states. Nature Communications, 16, 2065.

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Top 5 most cited protocols using «penicillin»

Monocyte differentiation and activation

Cited 103 times

PBMC (1 × 10⁶/mL) are cultured in RPMI 1640 supplemented with 10% human serum, 2 mM L-glutamine, and 1% penicillin (Invitrogen Ltd, Paisley, UK) and incubated at 37°C in a humidified 5% CO₂ atmosphere for 2 h in a 12-well plate. After 2 h, non-adhering PBMCs are harvested and discarded; monocytes (adhering cells) are culture in medium alone (unstimulated) or primed with 2 μg/mL LPS for 2 h (Sigma–Aldrich, St. Louis, MO) before stimulation with Nigericine (5 μM) (Sigma–Aldrich) for 1 h at 37°C in a humidified 5% CO₂ atmosphere. Adhering cells (monocytes) are then collected by trypsin treatment and prepared for FlowSight analysis by immunofluorescence staining as THP1-derived macrophage (see above).

Cossarizza A., Chang H.D., Radbruch A., Acs A., Adam D., Adam-Klages S., Agace W.W., Aghaeepour N., Akdis M., Allez M., Almeida L.N., Alvisi G., Anderson G., Andrä I., Annunziato F., Anselmo A., Bacher P., Baldari C.T., Bari S., Barnaba V., Barros-Martins J., Battistini L., Bauer W., Baumgart S., Baumgarth N., Baumjohann D., Baying B., Bebawy M., Becher B., Beisker W., Benes V., Beyaert R., Blanco A., Boardman D.A., Bogdan C., Borger J.G., Borsellino G., Boulais P.E., Bradford J.A., Brenner D., Brinkman R.R., Brooks A.E., Busch D.H., Büscher M., Bushnell T.P., Calzetti F., Cameron G., Cammarata I., Cao X., Cardell S.L., Casola S., Cassatella M.A., Cavani A., Celada A., Chatenoud L., Chattopadhyay P.K., Chow S., Christakou E., Čičin-Šain L., Clerici M., Colombo F.S., Cook L., Cooke A., Cooper A.M., Corbett A.J., Cosma A., Cosmi L., Coulie P.G., Cumano A., Cvetkovic L., Dang V.D., Dang-Heine C., Davey M.S., Davies D., De Biasi S., Del Zotto G., Cruz G.V., Delacher M., Bella S.D., Dellabona P., Deniz G., Dessing M., Di Santo J.P., Diefenbach A., Dieli F., Dolf A., Dörner T., Dress R.J., Dudziak D., Dustin M., Dutertre C.A., Ebner F., Eckle S.B., Edinger M., Eede P., Ehrhardt G.R., Eich M., Engel P., Engelhardt B., Erdei A., Esser C., Everts B., Evrard M., Falk C.S., Fehniger T.A., Felipo-Benavent M., Ferry H., Feuerer M., Filby A., Filkor K., Fillatreau S., Follo M., Förster I., Foster J., Foulds G.A., Frehse B., Frenette P.S., Frischbutter S., Fritzsche W., Galbraith D.W., Gangaev A., Garbi N., Gaudilliere B., Gazzinelli R.T., Geginat J., Gerner W., Gherardin N.A., Ghoreschi K., Gibellini L., Ginhoux F., Goda K., Godfrey D.I., Goettlinger C., González-Navajas J.M., Goodyear C.S., Gori A., Grogan J.L., Grummitt D., Grützkau A., Haftmann C., Hahn J., Hammad H., Hämmerling G., Hansmann L., Hansson G., Harpur C.M., Hartmann S., Hauser A., Hauser A.E., Haviland D.L., Hedley D., Hernández D.C., Herrera G., Herrmann M., Hess C., Höfer T., Hoffmann P., Hogquist K., Holland T., Höllt T., Holmdahl R., Hombrink P., Houston J.P., Hoyer B.F., Huang B., Huang F.P., Huber J.E., Huehn J., Hundemer M., Hunter C.A., Hwang W.Y., Iannone A., Ingelfinger F., Ivison S.M., Jäck H.M., Jani P.K., Jávega B., Jonjic S., Kaiser T., Kalina T., Kamradt T., Kaufmann S.H., Keller B., Ketelaars S.L., Khalilnezhad A., Khan S., Kisielow J., Klenerman P., Knopf J., Koay H.F., Kobow K., Kolls J.K., Kong W.T., Kopf M., Korn T., Kriegsmann K., Kristyanto H., Kroneis T., Krueger A., Kühne J., Kukat C., Kunkel D., Kunze-Schumacher H., Kurosaki T., Kurts C., Kvistborg P., Kwok I., Landry J., Lantz O., Lanuti P., LaRosa F., Lehuen A., LeibundGut-Landmann S., Leipold M.D., Leung L.Y., Levings M.K., Lino A.C., Liotta F., Litwin V., Liu Y., Ljunggren H.G., Lohoff M., Lombardi G., Lopez L., López-Botet M., Lovett-Racke A.E., Lubberts E., Luche H., Ludewig B., Lugli E., Lunemann S., Maecker H.T., Maggi L., Maguire O., Mair F., Mair K.H., Mantovani A., Manz R.A., Marshall A.J., Martínez-Romero A., Martrus G., Marventano I., Maslinski W., Matarese G., Mattioli A.V., Maueröder C., Mazzoni A., McCluskey J., McGrath M., McGuire H.M., McInnes I.B., Mei H.E., Melchers F., Melzer S., Mielenz D., Miller S.D., Mills K.H., Minderman H., Mjösberg J., Moore J., Moran B., Moretta L., Mosmann T.R., Müller S., Multhoff G., Muñoz L.E., Münz C., Nakayama T., Nasi M., Neumann K., Ng L.G., Niedobitek A., Nourshargh S., Núñez G., O’Connor J.E., Ochel A., Oja A., Ordonez D., Orfao A., Orlowski-Oliver E., Ouyang W., Oxenius A., Palankar R., Panse I., Pattanapanyasat K., Paulsen M., Pavlinic D., Penter L., Peterson P., Peth C., Petriz J., Piancone F., Pickl W.F., Piconese S., Pinti M., Pockley A.G., Podolska M.J., Poon Z., Pracht K., Prinz I., Pucillo C.E., Quataert S.A., Quatrini L., Quinn K.M., Radbruch H., Radstake T.R., Rahmig S., Rahn H.P., Rajwa B., Ravichandran G., Raz Y., Rebhahn J.A., Recktenwald D., Reimer D., e Sousa C.R., Remmerswaal E.B., Richter L., Rico L.G., Riddell A., Rieger A.M., Robinson J.P., Romagnani C., Rubartelli A., Ruland J., Saalmüller A., Saeys Y., Saito T., Sakaguchi S., de-Oyanguren F.S., Samstag Y., Sanderson S., Sandrock I., Santoni A., Sanz R.B., Saresella M., Sautes-Fridman C., Sawitzki B., Schadt L., Scheffold A., Scherer H.U., Schiemann M., Schildberg F.A., Schimisky E., Schlitzer A., Schlosser J., Schmid S., Schmitt S., Schober K., Schraivogel D., Schuh W., Schüler T., Schulte R., Schulz A.R., Schulz S.R., Scottá C., Scott-Algara D., Sester D.P., Shankey T.V., Silva-Santos B., Simon A.K., Sitnik K.M., Sozzani S., Speiser D.E., Spidlen J., Stahlberg A., Stall A.M., Stanley N., Stark R., Stehle C., Steinmetz T., Stockinger H., Takahama Y., Takeda K., Tan L., Tárnok A., Tiegs G., Toldi G., Tornack J., Traggiai E., Trebak M., Tree T.I., Trotter J., Trowsdale J., Tsoumakidou M., Ulrich H., Urbanczyk S., van de Veen W., van den Broek M., van der Pol E., Van Gassen S., Van Isterdael G., van Lier R.A., Veldhoen M., Vento-Asturias S., Vieira P., Voehringer D., Volk H.D., von Borstel A., von Volkmann K., Waisman A., Walker R.V., Wallace P.K., Wang S.A., Wang X.M., Ward M.D., Ward-Hartstonge K.A., Warnatz K., Warnes G., Warth S., Waskow C., Watson J.V., Watzl C., Wegener L., Weisenburger T., Wiedemann A., Wienands J., Wilharm A., Wilkinson R.J., Willimsky G., Wing J.B., Winkelmann R., Winkler T.H., Wirz O.F., Wong A., Wurst P., Yang J.H., Yang J., Yazdanbakhsh M., Yu L., Yue A., Zhang H., Zhao Y., Ziegler S.M., Zielinski C., Zimmermann J, & Zychlinsky A. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology, 49(10), 1457-1973.

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Corresponding organizations : University of Modena and Reggio Emilia, German Rheumatism Research Centre, Leibniz Association, Friedrich-Alexander-Universität Erlangen-Nürnberg, Kiel University, Technical University of Denmark, Lund University, Stanford University, University of Zurich, Inserm, Université Paris Cité, Assistance Publique – Hôpitaux de Paris, Hôpital Saint-Louis, University of Lübeck, Humanitas University, University of Birmingham, Technical University of Munich, University of Florence, University of Siena, National Cancer Centre Singapore, Duke-NUS Medical School, Sapienza University of Rome, Italian Institute of Technology, Istituto Pasteur, Medizinische Hochschule Hannover, Istituti di Ricovero e Cura a Carattere Scientifico, Fondazione Santa Lucia, Medical University of Vienna, University of California, Davis, Ludwig-Maximilians-Universität München, Urologische Klinik München, European Molecular Biology Laboratory, University of Technology Sydney, Helmholtz Zentrum München, Ghent University, VIB-UGent Center for Inflammation Research, University College Dublin, University of British Columbia, British Columbia Children's Hospital, Universitätsklinikum Erlangen, Monash University, Albert Einstein College of Medicine, California Institute for Regenerative Medicine, Thermo Fisher Scientific (United States), Luxembourg Institute of Health, University of Southern Denmark, Odense University Hospital, University of Luxembourg, Terry Fox Research Institute, University of Auckland, Maurice Wilkins Centre, German Center for Infection Research, Institute for Advanced Study, University of Rochester Medical Center, University of Verona, Peter Doherty Institute, University of Melbourne, Nankai University, University of Gothenburg, FIRC Institute of Molecular Oncology, Universitat de Barcelona, Délégation Paris 5, NYU Langone Health, Princess Margaret Cancer Centre, King's College London, National Health Service, Helmholtz Centre for Infection Research, Don Carlo Gnocchi Foundation, University of Milan, University of Milano-Bicocca, University of Cambridge, University of Leicester, de Duve Institute, Institut Pasteur, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Australian Regenerative Medicine Institute, Australian Research Council, The Francis Crick Institute, Istituto Giannina Gaslini, Novo Nordisk Foundation, University of Copenhagen, University Hospital Regensburg, University of Regensburg, Vita-Salute San Raffaele University, Istanbul University, Sony (United Kingdom), Charité - Universitätsmedizin Berlin, University of Palermo, University of Bonn, Agency for Science, Technology and Research, Singapore Immunology Network, University of Oxford, Freie Universität Berlin, Humboldt-Universität zu Berlin, University of Toronto, Heidelberg Institute for Stem Cell Technology and Experimental Medicine, University of Bern, Eötvös Loránd University, Leibniz Institute of Environmental Medicine, Leiden University Medical Center, Washington University in St. Louis, Centro de Investigacion Principe Felipe, Newcastle University, Institut Necker Enfants Malades, Centre National de la Recherche Scientifique, Hôpital Necker-Enfants Malades, Sorbonne Paris Cité, University of Freiburg, University Medical Center Freiburg, Owl Biomedical (United States), Nottingham Trent University, Leibniz Institute of Photonic Technology, University of Arizona, Henan University, The Netherlands Cancer Institute, Fundação Oswaldo Cruz, University of Massachusetts Chan Medical School, Istituto Nazionale Genetica Molecolare, University of Veterinary Medicine Vienna, SingHealth Duke-NUS Academic Medical Centre, Shanghai Jiao Tong University, University of California, Los Angeles, The University of Tokyo, Wuhan University of Technology, University of Cologne, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, University of Glasgow, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, German Cancer Research Center, German Cancer Society, Karolinska University Hospital, Murdoch Children's Research Institute, Houston Methodist, Universitat de València, INCLIVA Health Research Institute, MRC Epidemiology Unit, University Hospital of Basel, University of Minnesota, Delft University of Technology, Karolinska Institutet, Amsterdam University Medical Centers, University of Amsterdam, Sanquin, New Mexico State University, University Hospital Schleswig-Holstein, Chinese Academy of Medical Sciences & Peking Union Medical College, Shenzhen University, Shenzhen Institutes of Advanced Technology, University of Pennsylvania, Singapore General Hospital, Max Planck Institute for Infection Biology, University of Rijeka, Charles University, Jena University Hospital, National University of Singapore, ETH Zurich, Tulane University, Medical University of Graz, Goethe University Frankfurt, Max Planck Institute for Biology of Ageing, Berlin-Brandenburger Centrum für Regenerative Therapien, Osaka University, Nanyang Technological University, Université Paris Sciences et Lettres, Institut Curie, University of Chieti-Pescara, Institut Cochin, Caprion (Canada), Philipps University of Marburg, Beckman Coulter Foundation, Pompeu Fabra University, The Ohio State University, Erasmus MC, Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Université, Kantonsspital St. Gallen, Leibniz Institute of Virology (LIV), Roswell Park Comprehensive Cancer Center, Fred Hutch Cancer Center, IRCCS Humanitas Research Hospital, William Harvey Research Institute, University of Manitoba, National Institute of Geriatrics, Rheumatology and Rehabilitation, Institute for Experimental Endocrinology and Oncology, University of Sydney, Leipzig University, Northwestern University, Trinity College Dublin, Linköping University, Bambino Gesù Children's Hospital, Helmholtz Centre for Environmental Research, Klinikum rechts der Isar, Chiba University, University Medical Center Hamburg-Eppendorf, Universität Hamburg, Queen Mary University of London, University of Michigan–Ann Arbor, Centro de Investigación Biomédica en Red de Cáncer, Centro de Investigación del Cáncer, Instituto de Investigación Biomédica de Salamanca, Universidad de Salamanca, Burnet Institute, Amgen (United States), Universitätsmedizin Greifswald, Siriraj Hospital, Mahidol University, University of Tartu, Josep Carreras Leukaemia Research Institute, Universitat Autònoma de Barcelona, University of Udine, RMIT University, Utrecht University, University Medical Center Utrecht, Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Max Delbrück Center, Purdue University West Lafayette, Groene Hart Ziekenhuis, University of Alberta, Ospedale Policlinico San Martino, RIKEN Center for Integrative Medical Sciences, University of Lausanne, Istituto Neurologico Mediterraneo, Centre de Recherche des Cordeliers, University Hospital Bonn, Otto-von-Guericke University Magdeburg, Translational Research Institute, University of Lisbon, University of Brescia, Ashland (United States), BD Biosciences (United States), National Institutes of Health, Fraunhofer Institute for Cell Therapy and Immunology, Tsinghua University, Novartis (Switzerland), Pennsylvania State University, Alexander Fleming Biomedical Sciences Research Center, Universidade de São Paulo, CK-CARE, University Medical Center of the Johannes Gutenberg University Mainz, Johannes Gutenberg University Mainz, Babraham Institute, The University of Texas MD Anderson Cancer Center, Westmead Institute for Medical Research, University College London, University of London, Friedrich Schiller University Jena, TU Dortmund University, Leibniz Research Centre for Working Environment and Human Factors, University of Göttingen, Imperial College London, Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Simon Fraser University, Sichuan University, West China Hospital of Sichuan University, University Hospital of Bern

Neural Induction of Pluripotent Stem Cells

Cited 65 times

For dissociating intact colonies of pluripotent stem cells from the layer of DR4 feeders, hiPSCs were exposed to a low concentration of dispase (Invitrogen: 17105-041; 0.7 mg/ml) for ~30 min. Suspended colonies were subsequently transferred into ultra-low-attachment 100 mm plastic plates (Corning) in hiPSC medium without FGF2. For the first 24 h (day 0), the medium was supplemented with the ROCK inhibitor Y-27632 (EMD Chemicals). For neural induction, dorsomorphin (also known as compound C; Sigma 10 μM) and SB-431542 (Tocris, 10 μM) were added to the medium for the first five days. On the sixth day in suspension, the floating spheroids were moved to neural medium (NM) containing Neurobasal (Invitrogen: 10888), B-27 serum substitute without vitamin A (Invitrogen: 12587), GlutaMax (Invitrogen, 1:100), 100 U/ml penicillin and 100 μl streptomycin (Invitrogen). The NM was supplemented with 20 ng/ml FGF2 (R&D Systems) and 20 ng/ml EGF (R&D Systems) for 19 days with daily medium change in the first 10 days, and every other day for the subsequent 9 days. To promote differentiation of the neural progenitors into neurons, FGF2 and EGF were replaced with 20 ng/ml BDNF (Peprotech) and 20 ng/ml NT3 (Peprotech) starting at day 25, while from day 43 onwards only NM without growth factors was used for medium changes every four days.

Paşca A.M., Sloan S.A., Clarke L.E., Tian Y., Makinson C.D., Huber N., Kim C.H., Park J.Y., O’Rourke N.A., Nguyen K.D., Smith S.J., Huguenard J.R., Geschwind D.H., Barres B.A, & Paşca S.P. (2015). Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods, 12(7), 671-678.

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Corresponding organizations : Stanford University, University of California, Los Angeles, Yonsei University, Stanford Blood Center

SARS-CoV-2 Neutralization Assay Using Vero E6 Cells

Cited 59 times

Vero E6 cells were seeded at a density of 20,000 cells per well in a 96-well cell culture plate in complete Dulbecco’s Modified Eagle Medium (cDMEM). The following day, heat-inactivated serum samples (dilution of 1:10) were serially diluted threefold in 1× MEM (10% 10× minimal essential medium (Gibco), 2 mM l-glutamine, 0.1% sodium bicarbonate (wt/vol; Gibco), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Gibco), 100 U ml⁻¹ penicillin, 100 ug/ml⁻¹ streptomycin (Gibco) and 0.2% bovine serum albumin (MP Biomedicals)). The authentic SARS-CoV-2 virus (USA-WA1/2020; GenBank: MT020880) was diluted to a concentration of 100 TCID₅₀ (50% tissue culture infectious dose) in 1× MEM. Then, 80 μl of each serum dilution and 80 μl of the virus dilution were added to a 96-well cell culture plate and allowed to incubate for 1 h at room temperature. cDMEM was removed from Vero E6 cells and 120 μl of the virus–serum mixture was added to the cells. Then, the cells were incubated at 37 °C for 1 h. After the 1-h incubation, the virus–serum mixture was removed from the cells and 100 μl of each corresponding serum dilution and 100 μl of 1× MEM containing 1% FBS (Corning) was added to the cells. The cells were incubated for 48 h at 37 °C and then fixed with 10% paraformaldehyde (Polysciences) for 24 h at 4 °C. Following fixation, the paraformaldehyde was removed and the cells were washed with 200 μl PBS. The cells were then permeabilized by the addition of 150 μl PBS containing 0.1% Triton X-100 for 15 min at room temperature. The plates were then washed three times with PBS containing PBST and blocked in blocking solution (3% milk (American Bio) in PBST) for 1 h at room temperature. After blocking, 100 μl of mAb 1C7 (anti-SARS nucleoprotein antibody generated in house) at a dilution of 1:1,000 was added to all wells and the plates were allowed to incubate for 1 h at room temperature. The plates were then washed three times with PBST before the addition of goat anti-mouse IgG–HRP (Rockland Immunochemicals), diluted 1:3,000 in blocking solution for 1 h at room temperature. Plates were then washed three times with PBST, and SIGMAFAST OPD (Sigma–Aldrich) was added. After a 10-min incubation at room temperature, the reaction was stopped by adding 50 μl 3 M hydrochloric acid to the mixture. The OD490 was measured on a Synergy 4 plate reader (BioTek). A cut-off value of the average of the optical density values of blank wells plus three standard deviations was established for each plate and used to calculate the microneutralization titer. Microneutralization assays were performed in a facility with a biosafety level of 3.

Amanat F., Stadlbauer D., Strohmeier S., Nguyen T.H., Chromikova V., McMahon M., Jiang K., Arunkumar G.A., Jurczyszak D., Polanco J., Bermudez-Gonzalez M., Kleiner G., Aydillo T., Miorin L., Fierer D.S., Lugo L.A., Kojic E.M., Stoever J., Liu S.T., Cunningham-Rundles C., Felgner P.L., Moran T., García-Sastre A., Caplivski D., Cheng A.C., Kedzierska K., Vapalahti O., Hepojoki J.M., Simon V, & Krammer F. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature medicine, 26(7), 1033-1036.

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Corresponding organizations : Icahn School of Medicine at Mount Sinai, Peter Doherty Institute, University of Melbourne, University of California, Irvine, Alfred Health, Monash University, University of Helsinki

Measuring Cellular Oxygen Uptake Rates

Cited 36 times

Cells were grown in RPMI 1640 or MEM media (Invitrogen) with 10 % FBS (Atlanta Biologicals, Lawrenceville, GA) and supplemented with penicillin (85 U mL^-1) and streptomycin (85 μg mL^-1, Invitrogen). Typically cells in the log phase of growth were harvested by detachment with trypsin-EDTA (Invitrogen, Grand Island, NY) and washed 2 times by centrifugation at 300 g through HBSS. A Z2™ Coulter Counter^® was used to determine cell size distributions from the washed cells. The cell volumes reported are the nominal cell volumes. Cell diameters are estimated assuming a spheroid cell volume, 4/3 πr³. Cell counting was done with a Z2™ Coulter Counter^® in conjunction with a hemocytometer for confirmation. Care was taken to ensure that cellular debris did not produce a false over count and that cells were not sticking together to produce an undercount. For experiments using the Seahorse Bioscience XF96 instrument, cells were seeded between 5,000 and 100,000 cells well^-1; typical densities were between 15,000 - 30,000 cells per well; cell counts in the wells of the cell culture plate were verified after OCR determinations.
The rate of cellular oxygen uptake was monitored with an ESA BioStat Multi Electrode System (ESA Products, Dionex Corp, Chelmsford, MA) in conjunction with a YSI Oxygen Probe (5331) and glass reaction chamber vials in a YSI bath assembly (5301) (Yellow Springs Instruments, Yellow Springs, OH) all at room temperature. Cells were suspended in HBSS media (Invitrogen, Grand Island, NY) at a density of (3 − 30 × 10⁶) cells mL^-1; typical sample size was 2.00 mL. Cellular oxygen utilization was also determined using a Seahorse Bioscience XF96 extracellular flux analyzer (North Billerica, MA, USA). Cells were seeded into XF96 cell culture plates 24 or 48 h before experiments. OCR was determined using standard approaches for this technology [32 (link), 33 (link), 34 (link)], using XF96 FluxPaks (37 °C) from Seahorse Bioscience; Typically, Seahorse MEM media with 25 mM glucose and 1 mM sodium pyruvate was used.
Protein content of trypsinized cells was determined by the SDS-Lowry protein assay, using albumin from bovine serum (Sigma Chemical Co.; Cohn Fraction V, Sigma-A2153) as a standard [35 (link)].

Wagner B.A., Venkataraman S, & Buettner G.R. (2011). The Rate of Oxygen Utilization by Cells. Free radical biology & medicine, 51(3), 700-712.

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Corresponding organizations : University of Iowa

Feeder-Free, Serum-Free Pluripotent Stem Cell Culture

Cited 32 times

PSC were cultured at 37°C with 5% CO2. To achieve feeder-free, serum-free PSC culture, PSC were cultured on Synthemax™ plates^. (Corning). PSC were maintained in 2 mL mTeSR™-1 medium (Stem Cell Technologies) and passaged when colonies covered approximately 80% of the culture surface. To passage cells, the media was removed, PBS was added to wash cells, and 1 mL Collagenase IV (1 mg/mL, 200 U, Life Technologies, Invitrogen) was added and incubated for 4 min at 37°C. Next, the Collagenase IV was removed, cells were washed with PBS and 2 mL of warm mTeSR™-1 medium was added. To lift the cells, the cells were gently scraped and pipetted up and down to break up clumps to pieces 0.5–1 mm (not single cells) which were plated at ∼10,000 cells/cm² (corresponding to ∼1∶4 split). The passaged PSC was returned to incubator and not fed for the first 48 hours, but thereafter fed daily with fed 2 mL media (full media change). Alternatively, PSC were cultured on Mitomycin C-inactivated mouse embryo fibroblasts (MEFs) in PSC media. PSC media consisted of knock-out DMEM (Invitrogen), 10% knock-out-Serum Replacement (Invitrogen), 2 mM Glutamax-I (Gibco), 100 U/mL penicillin (Invitrogen), 100 µg/mL streptomycin (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.055 mM β-mercaptoethanol (R&D), 10 ng/mL bFGF (R&D), 5 µg/mL heparin (Sigma) and 0.5% Albumin (Sigma). MEF’s were prepared the day before on 0.1% gelatin-coated tissue culture plates (Corning) at a density of 50,000 cells/cm² in PSC media. The following day, PSC were plated at a density of 5,000–10,000 cells/cm² and supplemented with 1 mM Rock-inhibitor (Y27632; Calbiochem). Medium was changed daily by removing 50% of the media and replacing with fresh PSC medium. Colonies were passaged every 5 days by manual micro-dissection.

van Wilgenburg B., Browne C., Vowles J, & Cowley S.A. (2013). Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions. PLoS ONE, 8(8), e71098.

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Corresponding organizations : University of Oxford

Spelling variants (same manufacturer)

Penicillin-streptomycin-glutamine - 1552 citations Bocillin FL penicillin - 9 citations Streptomycin/D-penicillin - 5 citations Penicillins-streptomycin - 2 citations Dulbecco’s modified Eagle’s medium (DMEM), 100x penicillin/streptomycin (pen-strep) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) - 2 citations Penicillin-streptomycin (PNS) - 2 citations

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The spelling variants listed above correspond to different ways the product may be referred to in scientific literature.
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