Penicilline streptomycin

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Penicillin/streptomycin is a commonly used antibiotic combination in cell culture media. It provides broad-spectrum antimicrobial protection against a variety of bacteria.

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10 protocols using penicilline streptomycin

Breast Cancer Cell Line Cultivation

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For control experiments two different breast cancer cell lines, MCF7 and SKBR3 (both obtained from the ATCC; American Type Culture Collection, USA), were used. The MCF7 mammary adenocarcinoma cells were cultured in (v/v) 1:1 Dulbecco's Modified Eagle Medium (DMEM)/Ham's F12 medium (GIBCO-BRL Co, MD, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO-BRL), 2mM L-glutamine (GIBCO-BRL) 30mM NaHCOB_3B, 16 ng/ml insulin and 50 mg/ml penicilline/streptomycin (GIBCO-BRL). SKBR3 cells were used as positive control for HER2 and EGFR expression. SKBR3 cells were cultured in McCoy's (GIBCO-BRL) enriched with 10% fetal bovine serum and 2mM L-glutamine supplemented with 50 mg/ml penicilline/streptomycin. Cells were maintained in a humidified atmosphere of 5% COB_2B-95% air.
Sub-cultivation for all cell lines was performed with 0.25% trypsin and 5mM EDTA. All experiments were performed during the logarithmic growth phase. 15-20 h before each experiment and cells were transferred to serum-starved medium containing only L-glutamine, NaHCOB_3B and antibiotics.

Agelaki S., Dragolia M., Markonanolaki H., Alkahtani S., Stournaras C., Georgoulias V, & Kallergi G. (2016). Phenotypic characterization of circulating tumor cells in triple negative breast cancer patients. Oncotarget, 8(3), 5309-5322.

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Cultivation and Maintenance of HeLa Cell Lines

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Cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC). HeLa cells (ECACC, cat. no. 93021013) were grown in Dulbecco’s modified Eagle Medium (DMEM; Invitrogen, cat. no. 21969-035) supplemented with 10% fetal bovine serum (Thermo Fisher; cat. no. 10270106), 1% Penicilline/Streptomycin (Thermo Fisher; cat. no. 15140122), 2 mM L-Glutamine (Thermo Fisher; cat. no. 25030024). HeLa S3 (ECACC, cat. no. 87110901) were grown in spinner flasks (Corning, cat. No. 4500-125) in MEM medium supplemented with 2 mM L-Glutamine, 1% Non Essential Amino Acids (NEAA) (Thermo Fisher, cat. no. 11140050), 1% Penicilline/Streptomycin and 10% fetal bovine serum (Thermo Fisher). Both cell types were maintained in an environment with 5% CO₂ at 37°C. HeLa cells were sub-cultured at 1:5 ratio every 3 to 4 days using 0.05% trypsin-EDTA (GIBCO, cat. no. 25300054), while HeLa S3 were counted daily using Neubauer chamber and maintained at the 200 000 – 500 000 cells/mL concentration by diluting the culture with growth media.

Mendel M., Delaney K., Pandey R.R., Chen K.M., Wenda J.M., Vågbø C.B., Steiner F.A., Homolka D, & Pillai R.S. (2021). Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing. Cell, 184(12), 3125-3142.e25.

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Focused Ultrasound-Mediated pPrestin Transfection

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SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium containing growth factor F12 (DMEM/F12) (Gibco, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco), 1% penicillinestreptomycin (Gibco) and 1.2 g/L NaHCO₃ at 37 ℃. One day before experiment, a total of 3✕10⁴ cells were seeded in a 24-well plate in 500 µL medium (DMEM with 10% FBS). For the experiment, a pPrestin-MBs (concentration: 2✕10⁸ MBs/mL; pPrestin: 7500 ng) solution was added to the medium. The cell dish was flipped upside down and was sonicated by 1-MHz focused US (cycle number: 50-10000, pulse repetition frequency: 0.5-5 Hz, duration: 1 min, acoustic pressure: 0.1-0.7 MPa) to facilitate the pPrestin transfection (Fig. 2C). Four hours after 1-MHz focused US exposure, cells were washed with DPBS (Dulbecco's Phosphate-Buffered Saline, Biological Industries, CT, USA) and cultured in fresh medium. The gene transfection rate was assessed by counting the number of green fluorescence (Venus) positive cells at 48 h after gene transfection by flow cytometry (FACScalibur, BD Biosciences, CA, USA). Cell viability was determined using the Alamar Blue indicator (AbDSerotec, Oxford, UK).

Wu C.Y., Fan C.H., Chiu N.H., Ho Y.J., Lin Y.C, & Yeh C.K. (2020). Targeted delivery of engineered auditory sensing protein for ultrasound neuromodulation in the brain. Theranostics, 10(8), 3546-3561.

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GABA Receptor Subunit Detection

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RA was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Muscimol (Tocris Bioscience, Bristol, UK) and dimethyl sulfoxide (Amresco Solon, Ohio, USA) was purchased respectively. Sodium pentobarbital (100 mg/2 ml) and diazepam (10 mg/2 ml) were respectively purchased through Hanlim Pharm. Co., Ltd. and Samjin Pharm (Seoul, Korea). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle medium, neurobasal A medium, Trypsin-EDTA and Penicilline-Streptomycin was purchased from GIBCO (Grand island, NY, USA). N-(ethoxycarbonyl methyl)-6-methyl quinolinium bromide (MQAE) and cytosine beta-D-arabinofuranoside was purchased from Sigma-Aldrich Co. Specific polyclonal antibodies on the GABA_A receptors subunits of the GAD_65/67 extracted from rabbits and anti-rabbit immunoglobulin G-horseradish peroxidase was purchased from Abcam Inc. in Cambridge, UK. Chemiluminescent HRP substrate was purchased from Millipore Corporation (Billerica, MA, USA).

Kwon Y.O., Hong J.T, & Oh K.W. (2016). Rosmarinic Acid Potentiates Pentobarbital-Induced Sleep Behaviors and Non-Rapid Eye Movement (NREM) Sleep through the Activation of GABAA-ergic Systems. Biomolecules & Therapeutics, 25(2), 105-111.

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Synthesis and Characterization of Functionalized Gold Nanoparticles

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Gold chloride trihydrate (HAuCl₄·3H₂O), formaldehyde (37 wt. %) and phenol were bought from Shanghai Titan Polytron Technologies Inc. (Shanghai, China). Pluronic F127 (Mw= 12,600, PEO106PPO70PEO106) was obtained from Acros Corp. In addition, hydrochloric acid (HCl, 37%), sulfuric acid (H₂SO₄, 98%), nitric acid (HNO₃, ≥97.2), folic acid (FA), anhydrous dimethyl sulfoxide (DMSO) and sodium borohydride (NaBH₄) were all purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). N-hydroxysuccinimide (NHS), bovine serum albumin (BSA, 99.9%) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Annexin V-FITC/PI Apoptosis Detection Kit, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), Cell Counting Kit (CCK-8) and Calcein-AM/PI Double Stain Kit were all bought from YEASEN Corporation (Shanghai, China). Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), Penicilline Streptomycin and Trypsine EDTA used for cell culture were all obtained from Gibco Life Technologies. All chemical reagents used here were of analytical grade without further purification. The double-distilled water (18.2 MΩ, Millipore Co., USA) was employed in all experiments. In addition, phosphate buffered solutions (PBS, 0.1 M) were acquired by using 0.1 M Na₂HPO₄ and 0.1 M KH₂PO₄.

Zhang A., Pan S., Zhang Y., Chang J., Cheng J., Huang Z., Li T., Zhang C., de la Fuentea J.M., Zhang Q, & Cui D. (2019). Carbon-gold hybrid nanoprobes for real-time imaging, photothermal/photodynamic and nanozyme oxidative therapy. Theranostics, 9(12), 3443-3458.

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Synthesis and Characterization of Multifunctional Nanoparticles

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CPT was purchased from Dalian Meilun Biotech Co., Ltd. The (4-nitrophenyl) formate was obtained from Shanghai Energy Chemical Co., Ltd., Shanghai, China. Pyropheophorbide (PPA) was purchased from Shanghai Dibai Chemical Technology Co., Ltd., Shanghai, China. The phenylalanine-phenylalanine-glycine tripeptide was obtained from Zhejiang Ontores Biotechnologies Co., Ltd., Hangzhou, China. A cellular ROS Detection assay kit (DCFH-DA) were obtained from Dalian Meilun Biotech Co., Ltd., Dalian, China. Cell culture media RPMI 1640, Dulbecco's modified Eagle's medium (DMEM), penicilline–streptomycin and fetal bovine serum were purchased from GIBCO, Carlsbad, USA. 3-(4,5-Dimthyl-2-thiazolyl)-2,5-dipphenyl-2H-terazolium bromide (MTT) and trypsin-EDTA were obtained from Sigma‒Aldrich, USA. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG_2K) was purchased from Shanghai Advanced Vehicle Technology Co., Ltd. Other chemicals and solvents used in the present study were analytical or HPLC grade.

Sun M., Jiang H., Liu T., Tan X., Jiang Q., Sun B., Zheng Y., Wang G., Wang Y., Cheng M., He Z, & Sun J. (2021). Structurally defined tandem-responsive nanoassemblies composed of dipeptide-based photosensitive derivatives and hypoxia-activated camptothecin prodrugs against primary and metastatic breast tumors. Acta Pharmaceutica Sinica. B, 12(2), 952-966.

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Synthesis and Characterization of Compound 17i

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Compound 17i was self-prepare. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and 3-(4,5-Dimthyl-2-thiazolyl)-2,5-dipphenyl-2H-terazolium bromide (MTT) were obtained from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Cell culture media RPMI 1640, penicilline-streptomycin, and fetal bovine serum were available from GIBCO, (Carlsbad, United States). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-2000] (DSPE-PEG₂₀₀₀) was purchased from Shanghai Advanced Vehicle Technology Co., Ltd. All other chemical components and solvents applied in this study are of analytical grade.

Li B., Zhang X, & Li J. (2022). Carrier-free supramolecular nanoassemblies of pure LSD1 inhibitor for effective anti-tumor therapy. Frontiers in Chemistry, 10, 1012882.

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Luciferase-expressing Lewis Lung Carcinoma Cell Line

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Luciferase-expressing Lewis Lung Carcinoma (LLC-Luc) cell line of C57BL/6 mouse origin was purchased from Caliper Lifesciences. LLC-Luc cells were cultured in DMEM (Gibco, Gent, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Gent, Belgium), 2 mM glutamine (Gibco, Gent, Belgium), 100 UI/ml penicilline/streptomycin (Gibco, Gent, Belgium), 1 mg/ml geneticin (Serva, Heidelberg, Germany) and maintained in a humidified incubator at 37°C and in a 5% CO₂ atmosphere. Two type of LEC were used in this study: HMVEC-dLy (Lonza, Braine-l'Alleud, Belgium) and Human telomerase-transfected dermal LECs (hTERT-HDLECs) [37] (link). LEC were cultured in EGM2-MV medium (Lonza, Braine-l'Alleud, Belgium) until confluence was reached.

Maertens L., Erpicum C., Detry B., Blacher S., Lenoir B., Carnet O., Péqueux C., Cataldo D., Lecomte J., Paupert J, & Noel A. (2014). Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis. PLoS ONE, 9(9), e106976.

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Cell Culture Methodology for COS1 and CHO/hIRc

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COS1 cells (ATCC) were maintained in a humidified atmosphere of 5% CO₂ at 37 °C and cultured in DMEM media (Invitrogen) supplemented with 10% FBS (Invitrogen), 1% penicillinestreptomycin (Invitrogen), 1% GlutaMax (Invitrogen), and 1% non-essential amino acids (Invitrogen). CHO cells overexpressing the human insulin receptor (CHO/hIRc) were a kind gift from Dr. Michael L. Tremblay. CHO/hIRc cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C and cultured in Ham's F-12 media (Corning) supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% non-essential amino acids. For insulin treatment, cells were serum starved for 16 h prior to experimentation.

Garcia F.J, & Carroll K.S. (2014). Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cells. European journal of medicinal chemistry, 0, 28-33.

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Cultivation of Human Lung and Immune Cells

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Human lung alveolar epithelial A549 cells and human monocyte/macrophage U937 cells (The European Collection of Authenticated Cell Cultures) were cultured in Roswell Park Memorial Institute 1640 medium; the culture medium was supplemented with foetal bovine serum 10% (Invitrogen, Paisley, UK), 2 mM L-glutamine (Invitrogen), and 100 U ml À1 penicillinestreptomycin (Invitrogen).

Zhao H., Chen Q., Huang H., Suen K.C., Alam A., Cui J., Ciechanowicz S., Ning J., Lu K., Takata M., Gu J, & Ma D. (2019). Osteopontin mediates necroptosis in lung injury after transplantation of ischaemic renal allografts in rats. British journal of anaesthesia, 123(4).

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