Penicillin streptomycin glutamine 100x

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Penicillin-streptomycin-glutamine (100X) is a widely used cell culture supplement. It is a concentrated solution that provides penicillin, streptomycin, and glutamine to cell culture media. This product is commonly used to prevent bacterial contamination and support cell growth in cell culture applications.

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9 protocols using penicillin streptomycin glutamine 100x

Cell Culture Protocol for Proteomics

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C3A, HEK293T and HCT-116 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in the recommended medium. Base media, Dulbecco’s minimum essential medium (DMEM) and Eagle’s minimum essential medium (MEM), were from Gibco supplemented with Penicillin-Streptomycin-Glutamine 100x (Thermo Fisher Scientific, Waltham, MA) and 10% Seradigm fetal bovine serum (FBS, VWR, Radnor, PA). Cells for proteomics experiments were harvested as described previously and cells for western blot analysis were harvested the same way.^{18 (link)}

Golkowski M., Perera G.K., Vidadala V.N., Ojo K.K., Van Voorhis W.C., Maly D.J, & Ong S.E. (2018). Kinome chemoproteomics characterization of pyrrolo[3,4-c]pyrazoles as potent and selective inhibitors of glycogen synthase kinase 3. Molecular omics, 14(1), 26-36.

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Protein Extraction and Analysis Protocol

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Synthesis reagents were purchased from Millipore Sigma and Lumoprobe. Pierce high capacity streptavidin agarose (Thermo Fisher, #20357). Halt protease inhibitor cocktail, EDTA-free 100x (Thermo Fisher, #87785). M-PER™ mammalian protein extraction reagent (Thermo Fisher, #78501). RPMI 1640 medium (Thermo Fisher, #11875085). Penicillin-Streptomycin-Glutamine 100x (Thermo Fisher, #10378016). Bolt™ LDS sample buffer 4x (Thermo Fisher, #B0007). Bolt™ 4–12% Bis-Tris Plus Gels (Thermo Fisher, #NW04125BOX). Blot™ MES SDS running buffer 20x (Thermo Fisher, #B0002). PageRuler™ Plus prestained protein ladder, 10–250 kDa (Thermo Fisher, #26619). Pierce™ silver stain kit (Thermo Fisher, #24612). Anti-mouse c-Myc (9E10) antibody (Santa Cruz Biotechnology, #sc-40). Anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-358914). SuperSignal™ West Dura extended duration substrate (Thermo Fisher, #34075). iBlot™ 2 Transfer Stacks (Thermo Fisher, #IB23002). DNase I reaction buffer (New England Biolabs, B0303S). DNase I (RNAse-free, New England Biolabs, #M030S). 96-well plates (Corning, #3694). 384-wells plates (Greiner Bio-One, #788076).

Shirey R.J., Hart J.R., Sridharan B., Novick S.J., Turner L.D., Zhou B., Nielsen A.L., Eubanks L.M., Ueno L., Hixon M.S., Lairson L.L., Spicer T.P., Scampavia L.D., Griffin P.R., Vogt P.K, & Janda K.D. (2021). Synthetic fluorescent MYC probe: Inhibitor binding site elucidation and development of a high-throughput screening assay. Bioorganic & medicinal chemistry, 42, 116246.

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Cell Culture Protocols for Tumor Studies

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The cell lines were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA), including murine B16-F10 melanoma cells (CRL-6475), murine Lewis lung carcinoma LLC (CRL-1642), murine colon adenocarcinoma cells MC38 (Kerafast ENH204-FP), and human foreskin fibroblasts (HFFs) (SCRC-1041). Murine melanoma B16-F10 labeled with luciferase (B16-F10-Luc) was generated as described previously.42 (link) These cell lines were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, New York, USA) and 1% penicillin-streptomycin-glutamine 100x (Thermo, Waltham, Massachusetts, USA). HFFs were used to culture T. gondii ΔGRA17 tachyzoites. Lymphocytes were grown in RPMI 1640 medium containing 10% of HI-FBS, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, 1% penicillin–streptomycin–glutamine, and 1× minimal essential medium non-essential amino acids. Lymphocytes were used for flow cytometry analysis of tumor-infiltrating T cells and the immune status of the injected and uninjected tumors. Cultures of all above-mentioned cell lines and T. gondii were maintained in a humidified atmosphere at 37°C in 5% CO₂.

Zhu Y.C., Elsheikha H.M., Wang J.H., Fang S., He J.J., Zhu X.Q, & Chen J. (2021). Synergy between Toxoplasma gondii type I ΔGRA17 immunotherapy and PD-L1 checkpoint inhibition triggers the regression of targeted and distal tumors. Journal for Immunotherapy of Cancer, 9(11), e002970.

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Isolation and Culture of Carcinoma-Associated Fibroblasts

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Freshly resected specimens were washed with sterile phosphate-buffered saline (PBS) (BOSTER, AR0030, CA) containing 10% penicillin-streptomycin-glutamine (100X) (Gibco, 10,378,016, Thermo Fisher Scientific Inc.) before being minced into 1-mm³ fragments. These fragments were cultured in DMEM medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (10,099,141, Gibco) at 37 °C with 5% CO₂. This medium was used for carcinoma-associated fibroblasts (CAFs) and NFs [22 (link)–24 (link)]. Cultured cells at passages 3–6 were used.

Wang L., Yang Y., Xiong X., Yu T., Wang X., Meng W., Wang H., Luo G, & Ge L. (2018). Oral lichen-planus-associated fibroblasts acquire myofibroblast characteristics and secrete pro-inflammatory cytokines in response to Porphyromonas gingivalis lipopolysaccharide stimulation. BMC Oral Health, 18, 197.

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Culturing Breast Cancer Cell Lines

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MCF-7 (provided by American Type Culture Collection, ATCC, Manassas, Virginia, USA), MDA-MB-436 (provided by University Clinic Ulm, Ulm, Germany), MDA-MB-453 (provided by University Clinic Ulm, Ulm, Germany), MDA-MB-468 (provided by University Clinic Ulm, Ulm, Germany), and ZR75-1 (provided by Experimental Pharmacology and Oncology, Berlin-Buch, Berlin, Germany) were cultured in DMEM with L-glutamine (Gibco/ThermoFisher Scientific, Waltham, MA, USA), 10% FBS (Pan Biotech, Aidenbach, Germany), 1.2% L-glutamine (Gibco/ThermoFisher Scientific), 1.0% Penicillin-Streptomycin-Glutamine (100×) (Gibco/ThermoFisher Scientific), 1.0% MEM NEAA (non essential amino acid) (Gibco/ThermoFisher Scientific), 0.1% human recombinant insulin (Gibco/ThermoFisher Scientific), and 0.1% hEGF (Sigma-Aldrich/Merck, St. Louis, MO, USA) (Table 1) [22 (link),23 (link)]. HCC-1937 cells (provided by ATCC) were cultured in RPMI 1640 medium with 15% FBS (Pan Biotech, Aidenbach, Germany) and 1% of Penicillin-Streptomycin-Glutamine (100X) (Gibco/ThermoFisher Scientific). Cells were cultured in a humid 5% CO₂ incubator at 37 °C and all cell lines were negative for mycoplasma, verified by PCR.

Malka M.M., Eberle J., Niedermayer K., Zlotos D.P, & Wiesmüller L. (2021). Dual PARP and RAD51 Inhibitory Drug Conjugates Show Synergistic and Selective Effects on Breast Cancer Cells. Biomolecules, 11(7), 981.

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Culturing Colorectal Cell Lines

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Human colorectal cancer cell lines HCT116, DLD-1, HT-29, SW480, SW620, SW48, and HCT15 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The normal colonic epithelial cell line NCM460 was obtained from ScienCell Research Laboratories (San Diego, CA, USA). The normal colonic epithelial cell line HCoEpiC was obtained from INCELL (San Antonio, TX, USA). All cells were maintained at 37℃, and 5% CO2 in RPMI-1640 (C11875500BT, GIBCO) supplemented with 10% fetal bovine serum (FBS) (10270106, GIBCO) and penicillin-streptomycin-glutamine (100X) (10378016, GIBCO).

Liu C., Wang L., Liu X., Tan Y., Tao L., Xiao Y., Deng P., Wang H., Deng Q., Lin Y., Jie H., Zhang H., Zhang J., Peng Y., Zhang H., Zhou Z., Sun Q., Cen X, & Zhao Y. (2021). Cytoplasmic SHMT2 drives the progression and metastasis of colorectal cancer by inhibiting β-catenin degradation. Theranostics, 11(6), 2966-2986.

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Long-Term Culture-Initiating Cell Assay

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CD34+ cells were sorted at limiting dilution directly into 96-well plates on a feeder layer of MS5 cells (DSMZ, ACC441) in Myelocult™ H5100 (Stem Cell Technologies) supplemented with 10^-6 M hydrocortisone (Stem Cell Technologies) and 1% v/v penicillin-streptomycin-glutamine (100X) (Gibco). Cells were cultured for 5 weeks with 10 μM 3DA or vehicle (DMSO), half-medium changes were performed weekly. After 5 weeks medium was replaced by Methocult H4335 (Stem Cell Technologies) and incubated for two further weeks at 37°C/5% CO2. Colony formation was assessed by phase-contrast microscopy after seven weeks. The LTC-IC frequency of each experiment was calculated with ELDA software ^{51 (link)}.

Guerrero A., Innes A.J., Roux P.F., Buisman S.C., Jung J., Ortet L., Moiseeva V., Wagner V., Robinson L., Ausema A., Potapova A., Perdiguero E., Weersing E., Aarts M., Martin N., Wuestefeld T., Muñoz-Cánoves P., de Haan G., Bischof O, & Gil J. (2022). 3-deazaadenosine (3DA) alleviates senescence to promote cellular fitness and cell therapy efficiency in mice. Nature aging, 2, 851-866.

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Expansion of Cord Blood HSPCs

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Cord blood-derived HSPCs were cultured in Stemspan (Stem Cell Technologies) supplemented with 1% v/v penicillin-streptomycin-glutamine (100X) (Gibco) and the cytokines FLT3L, TPO and SCF (R&D Systems), all at a concentration of 100ng/mL. The cells were seeded at a starting density of 250.000 cells/mL and treated with 10 μM 3DA or vehicle (DMSO) at days 1, 4, and 7. Cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C. Cells were harvested and counted manually with a hemocytometer and trypan blue at day 9. These cells were analyzed by FACS and used for RNA-seq and xenotransplantation. The percentage of CD34+CD38+ cells was determined after blocking with Fc block (BD) for 10 minutes at room temperature and incubation with CD34+PE-Cy7 (BD, 348811) and CD38+FITC (BD, 555459) for 20-25 minutes at 4°C in the dark. Afterward, cells were washed and resuspended with PBS+BSA 0.2% containing a viability dye (PI). Samples were analyzed on a FACSCanto II (BD).

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Isolation and Culture of Oral Fibroblasts

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Fresh specimens were washed three times in sterile phosphate-buffered saline (PBS) (Gibco) containing 5% penicillin-streptomycin-glutamine (100X) (Gibco) and minced into fragments. These fragments were cultured in DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics, at 37 °C with 5% CO2. OLP AFs and normal mucosal fibroblasts (NFs) were used for experiments at 3-6 generations.

Cheng J., Zhang Y., Yang J., Wang Y., Xu J, & Fan Y. (2022). MiR-155-5p modulates inflammatory phenotype of activated oral lichen-planus-associated-fibroblasts by targeting SOCS1. Molecular biology reports, 49(8).

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