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Penicillin streptomycin stock solution

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Penicillin/streptomycin stock solution is a commonly used antibiotic mixture for cell culture applications. It provides a combination of the antibiotics penicillin and streptomycin in a concentrated liquid form.

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14 protocols using penicillin streptomycin stock solution

1

Prostate Cancer Cell Culture Conditions

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LNCaP, C4–2, 22RV1, DU-145, and PC-3 cells were cultured in RPMI 1640 medium (ThermoFisher (Waltham, MA, USA)) supplemented with 10% fetal bovine serum (Corning (Glendale, AZ, USA), and 1% penicillin-streptomycin stock solution (ThermoFisher). LAPC4 cells were maintained in IMDM medium (ThermoFisher), also supplemented with fetal bovine serum and penicillin-streptomycin. NCI-H660 cells were maintained in Advanced DMEM/F-12 (ThermoFisher) supplemented with 1X B-27 supplement (ThermoFisher), 10 ng/ml FGF2 (Peprotech (Cranbury, NJ, USA)), 10 ng/ml EGF (Peprotech), 1X Glutamax (ThermoFisher) and 1X penicillin-streptomycin (ThermoFisher). LNCaP-CXCR2 cells were generated as previously described[13 ] and maintained in RPMI 1640 medium, supplemented with 400 μg/ml Geneticin (ThermoFisher), 10% fetal bovine serum, and 1% penicillin-streptomycin stock solution.
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2

SILAC Labeling of Breast Cancer Cells

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Human breast cancer cells lines MDA-MB-231, BT-549, and Hs578T were obtained from American Type Culture Collection (ATCC). HEK293T cells were obtained from Cell Line Services (CLS). Cells were cultured in Dulbecco's modified eagle's medium (DMEM, PAN, Aidenbach, Germany) supplemented with 10% fetal calf serum (PAN) and 1% penicillin/streptomycin stock solution (Gibco/Invitrogen, Paisley, UK) at 37°C in humidified air containing 5% CO2. For MS experiments, cells were cultured for two weeks in SILAC DMEM (without arginine, lysine and glutamine, high glucose [4.5 g/l]) supplemented with 10% fetal bovine serum and labeled or unlabeled arginine and lysine, respectively (Silantes, Munich, Germany).
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3

Plasmid Transfection and Cell Culture

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HEK293T cells(293T/17 [HEK 293T/17] (ATCC® CRL11268™)) were cultured in DMEM medium with supplement of 10% FBS (GIBCO) and 1% Penicillin-Streptomycin stock solution (Invitrogen, 5,000unit Penicillin and 5,000 μg Streptomycin per milliliter). The expression vectors of HA/V5-tagged A3B and A3DE were constructed in our laboratory. HA-tagged A3DE-E80Q, A3DE-E264Q, A3DE- E80Q/E264Q and A3B-W228L/ D316N were generated by overlapping PCR and confirmed by sequencing. The human L1 plasmids 99 PUR L1RP EGFP (L1) and 99 PUR JM111 EGFP (JM111, an L1 construct containing two point mutations in ORF1, which cause complete abolishment of LINE-1 retrotransposition, was used as a negative control) were gifted from Professor Kazazian HH Jr and had been described previously [40 (link)]. The pcDNA3.1-EGFP plasmid was cloned in our laboratory.
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4

Culturing Human Multiple Myeloma Cell Lines

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Human multiple myeloma cell lines LP‐1, OPM‐2, RPMI‐8226, and ANBL‐6 cells, and the human stromal cell line HS‐5, were obtained from the American Type Culture Collection (ATCC, Molsheim, France). The XG‐2 cell line was kindly provided by J Moreaux (University of Montpellier, Montpellier, France). Myeloma cell lines and stromal cells were cultured in RPMI‐1640 and DMEM medium (Lonza, Basel, Switzerland) respectively, supplemented with 2 mm l‐glutamine (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin stock solution (Thermo Fisher Scientific), and 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) at 37 °C in 5% CO2. ANBL‐6 and XG‐2 cells were supplemented with 2 ng/ml recombinant IL‐6 (R&D Systems, Abingdon, UK). The authenticity of the human multiple myeloma cell lines was regularly confirmed by short tandem repeat analysis. The cell lines were checked monthly for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza).
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5

HDL-c Isolation via KBr Ultracentrifugation

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HDL-c isolation with sequential potassium bromide (KBr) density ultracentrifugation as described [50 (link)]. To the plasma was added 1% antibiotics (penicillin/streptomycin stock solution, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), protease inhibitor cocktail (cOmpleteTM, Roche, Basel, Switzerland) to avoid ex vivo oxidation and degradation. Purified HDL-C was dialyzed against a degassed solution of Buffer A (20 mM Tris-HCl plus 0.5 mM EDTA) on a shaker at 4 °C with five buffer changes (once/day).
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6

Hypoxia-Reoxygenation Injury Model in Murine Renal Tubular Cells

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A murine renal tubular epithelial cell line (TEC; Fuheng Cell Center, Shanghai, China) was cultured in Dulbecco’s modified Eagle’s medium–nutrient mixture F-12 (HyClone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin stock solution (Thermo Fisher Scientific). To simulate I/R injury in vitro, we used a model of hypoxia/reoxygenation (H/R). TEC cells were exposed to a hypoxia incubator chamber (STEMCELL Technologies, Vancouver, BC, Canada) with an anoxic mixture gas (95% N2 and 5% CO2) for 12h at 37 °C. Then, cells were returned to complete growth medium and placed in a normoxic chamber (37 °C, 5% CO2) for 2 h of reoxygenation.
Lats2 was overexpressed via transfecting with a lentivirus incorporating the Lats2 recombinant plasmid (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, GENECHEM, Shanghai, China), referred to as LV-Lats2. LV-NC, a lentivirus incorporating an empty plasmid, served as a negative control. Viral particles were removed 16 h later after transfection, and cells were replenished with fresh TEC medium. Puromycin selection commenced 24 h later with the addition of 2 μg/mL puromycin. Surviving cells were isolated into single cell clones. The cells after treatment were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) for western blotting analysis.
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7

Xenopus Oocyte Preparation and Maintenance

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Stage VI X. laevis oocytes were obtained as previously described [71 (link)]. Oocytes were maintained in L-15 medium solution (Sigma-Aldrich Cat# L4386) supplemented with HEPES (Sigma-Aldrich Cat# H4034), 0.1% (v/v) of penicillin/streptomycin stock solution (ThermoFisher Scientific Cat# 15140–122) and 0.1% (v/v) of gentamycin (EMD Millipore Cat# 345814-1GM) at pH 7.6.
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8

Cytokine Release Assay for Immune Cells

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Cells were plated in flat-bottomed 24-well plates at a density of 2.5 x 106 cells (for RBC-lysed total bone marrow cells or total splenocytes) in 500 μl of complete cell medium, or in flat-bottomed 96-well plates at a density of 50,000 cells (for HSPC-derived macrophages) in 200 µl of complete cell culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin stock solution [Gibco]). Whole blood was diluted 1:2 in complete cell culture medium at a final volume of 200 μl and plated in flat-bottomed 96-well plates. To prevent potential fungal growth, 0.5 μg/ml amphotericin B was added to the cultures of total bone marrow cells, total splenocytes and blood from infected animals. Cells were challenged with 100 ng/ml of Pam3CSK4, 100 ng/ml of ultrapure Salmonella minnesota LPS (all from In vivogen) or 25 x 106 inactivated C. albicans ATCC 26555 yeasts for 24 h, and cell-free supernatants were then harvested and tested for cytokine release using commercial enzyme-linked immunosorbent assay (ELISA) kits [TNF-α and IL-6 (eBioscience)]. Unstimulated cells served as negative controls. Triplicate samples were analyzed in each assay.
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9

Isolation and Culture of Murine Splenocytes and Peritoneal Macrophages

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Splenocytes were obtained from control or Pam3CSK4-treated mice as described elsewhere (Murciano et al., 2007 (link); Yáñez et al., 2011 (link)). Briefly, total spleen cells were obtained by collagenase D treatment of the organ, erythrocytes were lysed using ammonium chloride buffer (BD FACS™ lysing solution), and cells were washed and counted.
Resident peritoneal macrophages were harvested by instilling and withdrawing 10 ml of complete cell culture medium: RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin stock solution (Gibco, Barcelona, Spain). Cells were washed once with the same medium, and plated at a density of 1.5 x 105 cells in 200 μl of medium per well, in a 96 well tissue culture plate. Peritoneal macrophages were allowed to adhere for 5 h at 37°C in a 5 % CO2 atmosphere, the non-adherent cells were removed, and the adherent macrophages were cultured for 72 h prior to be challenged with the indicated stimuli.
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10

Lipoprotein Isolation and Purification

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Lipoproteins were isolated with sequential potassium bromide density ultracentrifugation as described [42 (link)]. The plasma was obtained from freshly collected whole blood samples, and 1% antibiotics (penicillin/streptomycin stock solution, Gibco), 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), and a protease inhibitor cocktail (cOmplete, Roche, Sigma-Aldrich, St. Louis, MO, USA) were added to avoid ex vivo oxidation and degradation. LDL-C particles were isolated by using sequential potassium bromide density ultracentrifugation. Purified LDL-C was dialyzed against a degassed solution of 20 mM Tris-HCl and 0.5 mM EDTA at 4 °C with five buffer changes (once/day).
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