Penicillin streptamycin

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Penicillin/Streptamycin is a sterile liquid solution containing the antibiotics penicillin and streptomycin. It is commonly used as a supplement in cell culture media to prevent bacterial contamination.

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Penicillin (24825 citations) Penicillin-streptamycin-glutamine (3 citations) Penicillin and Streptamycin (2 citations)

8 protocols using penicillin streptamycin

Murine ES Cell Line Differentiation

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Murine ES cell lines described previously [57 (link)] were grown in high glucose DMEM with stable glutamine (GIBCO) containing 10 % FBS Superior (Biochrom), 100 μM non-essential amino acids (GIBCO), 1 % Penicillin/Streptamycin (GIBCO) and 100 μM β-Mercaptoethanol (Sigma) in presence of 1000 U/mL of Leukemia inhibitory factor (LIF, Milllipore). Differentiation of aCaBs was performed in hanging drop culture for two days using 1000 cells as starting material for one EB in Iscove’s basal medium (Biochrom) containing 10 % FBS (Biochrom), 100 μM non-essential amino acids (GIBCO), 1 % Penicillin/Streptamycin (GIBCO) and 450 μM 1-Thioglycerol. For additional 4 days, the cells were differentiated in suspension culture, and at day 6 of differentiation consistently 15 EBs were seeded on one well of a 24-well-plate. Antibiotic selection with 400 μg/mL G418 (Biochrom) was initiated at day 8 post seeding. 4 days thereafter, aCaBs were isolated via treatment with 6000 U/mL Collagenase IV (GIBCO) for 30 min. To obtain single cells for subsequent experiments, the bodies were further dissociated with 100 % Accutase (Affimetrix) for 15 min. To ensure successful generation of aCaBs, potential mycoplasma contamination was routinely controlled twice a week using the PCR based MycoSPY kit system (Biontex).

Wolfien M., Rimmbach C., Schmitz U., Jung J.J., Krebs S., Steinhoff G., David R, & Wolkenhauer O. (2016). TRAPLINE: a standardized and automated pipeline for RNA sequencing data analysis, evaluation and annotation. BMC Bioinformatics, 17, 21.

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Cell Line Cultivation and Maintenance

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SUM149 and SUM159 were purchased from Asterand Bioscience (Detroit, MI, USA). All other cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). BT474 cells were grown in Hybri-Care Media (ATCC); BT-549, HCC39, HCC70, MDA-MB-231, MDA-MB-453, MDA-MB-468, T47D, ZR-75-10 in RPMI 1640 (Corning, Corning, NY, USA); MCF7 and MDA-MB-453 in DMEM (Corning); and SK-BR-3 in McCoy’s 5A (Sigma-Aldrich, St. Louis, MI, USA). Media was supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA) and 1% penicillin–streptamycin (Gibco, Waltham, MA, USA). SUM149 and SUM159 cells were grown in Ham’s/F12 (Gibco), supplemented with 5% FBS, 1% penicillin–streptamycin, 1M HEPES (Gibco), and 1 mg/mL hydrocortisone (Sigma-Aldrich). All cell lines were maintained at 37 °C with 5% CO₂. All cell lines were confirmed to be free of mycoplasma contamination (Bimake, Houston, TX, USA or Lonza, Basal, Switzerland) every 2 months.

Cuellar-Vite L., Weber-Bonk K.L., Abdul-Karim F.W., Booth C.N, & Keri R.A. (2022). Focal Adhesion Kinase Provides a Collateral Vulnerability That Can Be Leveraged to Improve mTORC1 Inhibitor Efficacy. Cancers, 14(14), 3374.

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LCMV Cl13 Virus Propagation in BHK-21 Cells

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LCMV Cl13 virus was propagated using BHK-21 cells. BHK-21 cells were maintained in DMEM supplemented with 10% FBS, 1% Penicillin/Streptamycin (Gibco), 1% L-glutamine (Gibco), and 5% tryptose phosphate broth (Gibco). BHK-21 cells were infected with a MOI of 0.01 of LCMV Cl13 virus stocks (kind gift of Juan Carlos de la Torres) and gently rocked for 1 hour before removal of virus and replacement of media. After 48 hours of virus propagation, viral supernatant was collected, aliquoted and stored. Quantification of virus stocks was completed using the focus forming assay and Vero cells.

Hope J.L., Otero D.C., Bae E.A., Stairiker C.J., Palete A.B., Faso H.A., Lin M., Henriquez M.L., Roy S., Seo H., Lei X., Wang E.S., Chow S., Tinoco R., Daniels G.A., Yip K., Campos A.R., Yin J., Adams P.D., Rao A, & Bradley L.M. (2023). PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion. Cell reports, 42(5), 112436.

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Isolation and Characterization of Murine Embryonic Fibroblasts

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The Fancd2^+/− C57/BL6 sv129 hybrid background mice were timed mated, and murine embryonic fibroblasts (MEFs) isolated from pups at E13.5, confirmed by PCR [32 (link)] and transformed with a plasmid containing the SV40 large T antigen. Wild type, Fancd2^−/−, Parp1^+/+ or Parp1^−/− MEFs and human eHAP were grown in DMEM and IMDM medium supplemented with 10% FBS, Penicillin/streptamycin (Gibco) respectively. Patient-derived HSC72 (FANCA-deficient) and NV012 human cell lines were grown in RPMI1640 medium supplemented with 10% FBS, Penicillin/streptomycin (Gibco) and 50 μM 2-mercartoethanol (Merck). 5dC (D3897; Sigma), 5mdC (PY 7635; B&A), 5hmdC (PY 7588; B&A), 5fdC (PY 7589; B&A), 5cadC (PY 7593; B&A), BrdU (B5002; Sigma), CldU (Sigma, C6891), IdU (Sigma, I7125), EdU (Thermo Scientific, A10044) were dissolved in PBS (10 mM stock solution), and olaparib (Selleckchem, S1060) was dissolved in DMSO (10 mM stock solution), and added to cell cultures as indicated. The antibodies used were against ser139-H2AX (Millipore, 05-636), PAR (Millipore, MABE1016), FANCD2 (Novus, NB100-316), ser345-CHK1 (Cell Signaling 2348), CHK1 (sc-8408), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (AbSerotec, OBT0030), BrdU (BD Bioscience, 347580), α-tubulin (Sigma, T9026), ser4/ser8 RPA32 (Bethyl, A300-245A).

Peña-Gómez M.J., Moreno-Gordillo P., Narmontė M., García-Calderón C.B., Rukšėnaitė A., Klimašauskas S, & Rosado I.V. (2022). FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine. Cell Death & Disease, 13(5), 503.

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Isolation and Culture of Dorsal Root Ganglia

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Mice were euthanized with pentobarbital (300 mg/kg). L4, L5, L6 dorsal root ganglia were placed in cold PBS and stripped of axonal extension. Ganglia were enzymatically dissociated with type I collagenase (2 mg/mL, Gibco) and Dispase (5 mg/mL, Gibco) for 30 minutes at 37 °C and mechanically dissociated with a fire-polished Pasteur pipette. The cell suspension was centrifuged for 10 minutes at 800 g and re-suspended in MEMα medium (Gibco) supplemented with 10% horse serum (Life Technologies) and 1% penicillin-streptamycin (Gibco). Cells were then plated on poly-D-lysine (Sigma)-coated fluorodishes (WPI). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Experiments were performed 24 h after plating.

Lalisse S., Hua J., Lenoir M., Linck N., Rassendren F, & Ulmann L. (2018). Sensory neuronal P2RX4 receptors controls BDNF signaling in inflammatory pain. Scientific Reports, 8, 964.

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Murine Islet Isolation and Scaffold Seeding

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All murine studies were approved by the Northwestern University Animal Care and Use Committees. Male C57BL/6 mice (Jackson Laboratories, Bar Harbor ME) 8–12 weeks old were used as islet donors. Islet isolation was performed as previously described [41 ]. Briefly, donor mice were anesthetized with an intraperitoneal injection of 50 mg/kg Ketamine (Henry Schein)/Xylazine (Anased, Lloyd Labs). After a midline abdominal incision, the common bile duct was cannulated and injected with a cold solution of collagenase (type XI; Sigma Chemical Company, St Louis, MO) in Hank’s balanced salt solution. The pancreas was dissected, removed, and digested at 37°C for 15 min. After filtration through a mesh scre en, the filtrate was applied to a discontinuous Biocoll separating gradient (Cedarlane). Isolated islets were hand-counted and 70 islets were seeded on each scaffold using a fine glass pipette under a surgical microscope. Scaffolds were examined after seeding to ensure a seeding efficiency of >95%. Scaffolds were then incubated at 37°C in 5% CO₂ and 95% air for 20 min, before adding serum-free RPMI 1640, supplemented with 1% L-Glutamine and 1% Penicillin/Streptamycin, (Gibco), and used for transplantation.

Minardi S., Guo M., Zhang X, & Luo X. (2018). An elastin-based vasculogenic scaffold promotes marginal islet mass engraftment and function at an extrahepatic site. Journal of immunology and regenerative medicine, 3, 1-12.

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Cell Culture Protocols for Endothelial, Keratinocyte, and SCC Cells

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Normal human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVEC) were purchased from Lonza (Verviers, Belgium) and were grown in MCDB 131 medium (Gibco-Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 2mM glutamine, endothelial cell growth supplement (12 ng/ml; BD Biosciences, Bedford, MA) and 2.5 mg/ml heparin. Immortalized lymphatic endothelial cells (hTERT-LEC) were cultured in EGM2-MV (Lonza) supplemented with 5% fetal bovine serum and glutamine. Immortalized human keratinocytes (HaCaT cells) and three genital SCC cell lines (A431, HT-3, SiHa) were grown in a 3:1 mixture of D MEM (Gibco) and Ham's F12 (Gibco) containing 10% fetal calf serum (FCS) and supplied with 1% non-essential amino acid (Gibco), 1% sodium pyruvate (Gibco) and 0.5% penicillin-streptamycin (Gibco). Two head and neck SCC cell lines (Detroit 562, RPMI 2650) were maintained in MEM (Gibco) containing 10% FCS and supplemented with 1% L-glutamine (Gibco). All the cell lines were incubated until a 60–70% confluence was reached.

Suarez-Carmona M., Hubert P., Gonzalez A., Duray A., Roncarati P., Erpicum C., Boniver J., Castronovo V., Noel A., Saussez S., Peulen O., Delvenne P, & Herfs M. (2014). ΔNp63 isoform-mediated β-defensin family up-regulation is associated with (lymph)angiogenesis and poor prognosis in patients with squamous cell carcinoma. Oncotarget, 5(7), 1856-1868.

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LCMV Cl13 Virus Propagation in BHK-21 Cells

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