A549 cell line (ATCC: CCL-185) was maintained in DMEM medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies). H1299 (ATCC: CRL-5803) and CL1-5 [27 (link)] were maintained in RPMI medium (GibcoBRL Life Technologies) supplemented with 10% FBS and 1% penicillin-streptomycin-neomycin. All cells were cultured in a humidified incubator containing 5% CO2 at 37°C. Male SCID mice (C.B17/lcr-Prkdc scid/CrlNarl) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animal care and in vivo experiments were performed in compliance with the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee. Mice were provided a standard laboratory diet and distilled water and kept on a 12-h light/dark cycle at 25 ± 2°C and 55 ± 5% relative humidity.
Penicillin streptomycin neomycin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Switzerland, France
Penicillin-streptomycin-neomycin is a combination antibiotic solution used in cell culture media to prevent bacterial contamination. It contains the antibiotics penicillin, streptomycin, and neomycin.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (24 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (9 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (5 mentions)
Dmem medium, by Thermo Fisher Scientific (5 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (4 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Rpmi medium, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Sh sy5y, by Thermo Fisher Scientific (2 mentions)
U87 glioblastoma cell line, by American Type Culture Collection (2 mentions)
Sh sy5y, by American Type Culture Collection (2 mentions)
77 protocols using penicillin streptomycin neomycin
1
Cell Culture and Animal Maintenance Protocol
2
Induction of Chondrocyte Differentiation
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C5.18 (rat chondrocyte cell line) was kindly gifted by Dr Tong Weixue. These cells were maintained in complete alpha-minimum essential medium (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin–neomycin (complete culture medium; all from Invitrogen Corporation, Carlsbad, CA, USA) in a 5% CO2-humidified incubator at 37°C.
For induction by MMP13 or type X collagen (Col X), cells were transferred to serum-free DMEM (Dulbecco's Modified Eagle’s Medium, Invitrogen Corporation, Carlsbad, CA, USA) and then treated with 10 ng/mL IL-1β (R&D Systems, Minneapolis, MN, USA) and Rb1 at 100 μg/mL. In the IL-1β group, cells were treated with 10 ng/mL IL-1β alone. In the control group, they were untreated except for a change in the medium. Cells were harvested after incubation for 24 hours.
For induction by MMP13 or type X collagen (Col X), cells were transferred to serum-free DMEM (Dulbecco's Modified Eagle’s Medium, Invitrogen Corporation, Carlsbad, CA, USA) and then treated with 10 ng/mL IL-1β (R&D Systems, Minneapolis, MN, USA) and Rb1 at 100 μg/mL. In the IL-1β group, cells were treated with 10 ng/mL IL-1β alone. In the control group, they were untreated except for a change in the medium. Cells were harvested after incubation for 24 hours.
3
Cell Culture Conditions for Neuroscience Research
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The following cell lines were used: HEK-293 (ATCC CRL-1573); U87-glioblastoma cell line (ATCC HTB-14); SH-SY5Y (ATCC CRL-2266); 1321N1 human astrocytoma cell line (ECACC 86030402); LN-405 glioblastoma cell line (ACC 189). All cell cultures were maintained at 37°C in humidified atmosphere containing 5% CO2 and grown as monolayers in DMEM (Biochrom, Berlin, Germany), supplemented with 4.5 g/l glucose, 10% fetal bovine serum (Hyclone, Bonn, Germany), 1% penicillin/streptomycin/neomycin (Invitrogen, Karlsruhe, Germany). U87 and SH-SY5Y cells were grown in DMEM additionally supplemented with 1% non-essential amino acids (Invitrogen, Karlsruhe, Germany).
4
Isolation and Culture of Human Breast Cancer Fibroblasts
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Human breast cancer specimens were collected from the TMU Biobank (TMU-IRB, P102025, Taipei, Taiwan). Fresh breast cancer tissue was dissected using the aseptic technique, following which, the tissue was minced, trypsinized (addition of trypsin, a proteolytic enzyme that facilitates the breakdown of tissue into single cells), and seeded into T25 tissue culture flasks. The cells were grown at 37 °C under 5% CO2 in complete RPMI 1640 medium (Gibco, Amarillo, TX, USA), supplemented with 10% heat-inactivated FBS (Gibco, Amarillo, TX, USA) and 50 U/mL penicillin/streptomycin/neomycin (Invitrogen, Waltham, MA, USA) in a humidified (5% CO2, 37 °C) incubator. Fibroblast characteristics were confirmed via morphology and flow cytometry analysis with antibodies against Vimentin (ab20346, Abcam, Cambridge, UK) and alpha-smooth muscle actin (α-SMA, ab7817, Abcam, Cambridge, UK) [11 (link)].
5
Culturing Rat Nodose Ganglion Neurons
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Rat NG neurons were cultured as previously described [33] (link)–[34] (link). Briefly, the NG was dissected and dissociated from postnatal day 1 pups that were sacrificed by cervical dislocation under sterile conditions. The ganglia were then incubated for 15 min at 37°C in D-Hank’s balanced salt solution (without Ca2+ and Mg2+) with collagenase (type I, 1 mg/ml; Sigma). The cell suspension was then washed once with L-15 growth medium containing 10% horse serum. The pellet was resuspended and centrifuged through a Percoll (Pharmacia) gradient (35%) to separate neurons from non-neuronal cells. The neuronal suspension was then washed twice and plated on poly-D-lysine (0.1 mg/ml; Sigma-Aldrich)-coated glass coverslips (30 µg/ml in PBS overnight at 4°C) in Neurobasal-A medium (Invitrogen) supplemented with B-27 serum-free supplement (Invitrogen), 0.5 mM L-glutamine (Invitrogen), 2.5% fetal bovine serum (HyClone, Logan, UT), and 1% penicillin-streptomycin-neomycin (Invitrogen) in ultra violet-sterilized 24-well flat bottom plates (MaxiSorp, Nalge Nunc Int., Naperville, IL). They were then cultured for >10 days at 37°C in a humidified atmosphere of 5% CO2 and 95% air. For cultures grown longer than 3 days, the media were replaced with fresh medium on days 3 and 6.
6
Cytotoxic Effects of CoQ0 in Cell Cultures
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CoQ0 (2,3 dimethoxy-5-methyl-1,4 benzoquinone) was purchased from Sigma-Aldrich (St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), l -glutamine and penicillin/streptomycin/neomycin were obtained from GIBCO BRL/Invitrogen (Carlsbad, CA). p53, Bcl-2, and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc (Heidelberg, Germany). PARP antibody was obtained from Cell Signaling Technology, Inc (Danvers, MA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Chemical Co (St Louis, MO). CoQ0 was dissolved in dimethyl sulfoxide (DMSO) and diluted with DMEM to make the final concentration below 0.01%. All other chemicals were of the highest grade commercially available and were supplied either by Merck or Sigma.
7
In Vitro Neuroblastoma Cell Culture
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The mouse neuroblastoma cell line N2a is generally studied for neuronal differentiation research in vitro.24 , 25 N2a cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco 12100046, CA) supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Gibco A38401, CA), 50 U/mL penicillin/streptomycin/neomycin (Invitrogen 15640055, CA), nonessential amino acid solution (Thermo Fisher Scientific 11140050, CA) and sodium pyruvate (Sigma Aldrich P5280, MI) in a humidified (5% CO2, 37°C) incubator.
8
Culturing E0771-M Breast Tumor Cells
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The primary cultured lung-metastatic E0771 breast tumor cells (E0771-M) were harvested from the mouse E0771-allografted tumor. Tumor tissues were initially subjected to mechanical breakdown by mincing the tumors with a scalpel, followed by chemical digestion in 10 mL of 0.2% collagenase A (Sigma Aldrich 10103578001, Saint Louis, MO, USA) and 0.2% trypsin (Gibco 27250018, Amarillo, TX, USA), and 0.5% FBS in RPMI for 1 h at 37 °C. After digestion, tumor cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco, Amarillo, TX, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Amarillo, TX, USA) and 50 U/mL penicillin/streptomycin/neomycin (Invitrogen, Waltham, MA, USA), in a humidified (5% CO2, 37 °C) incubator.
9
Breast Cancer Cell Line and Tumor Sample Collection
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BT-549 (ATCC® HTB-122™), Hs 587T (ATCC® HTB-126™), MDA-MB-436 (ATCC® HTB-130™), MDA-MB-468 (ATCC® HTB-132™), MDA-MB-231 (ATCC® HTB-26™), MDA-MB-453 (ATCC® HTB- 131™), HCC1395 (ATCC® SC-CRL-2324™), HCC38 (ATCC® CRL-2314™), MCF 10A (ATCC® CRL-10317™), AU565 (ATCC® CRL-2341™), SK-BR-3 (ATCC® HTB-30™), BT-474 (ATCC® HTB-20™), HCC1419 (ATCC® CRL-2326™), HCC1954 (ATCC® CRL-2338™), T-47D (ATCC® HTB-133™), MCF7 (ATCC® HTB-22™) and ZR-75-1 (ATCC® CRL-1500™) cells were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). The cells were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/ F12, Gibco, California, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, California, USA) and 50 U/mL penicillin/ streptomycin/neomycin (Invitrogen, California, USA) in a humidified (5% CO2, 37°C) incubator. All human breast tumor samples (n = 234) were obtained as specimens from anonymous donors from Taipei Medical University Hospital, Taipei, Taiwan, as approved by the Institutional Review Board (IRB) and ethics committee of the institution (P102025). Histological inspection confirmed that all patient samples consisted of greater than 80% tumor tissue. All samples (each paired tumor tissue vs. normal tissue) were collected and categorized according to clinical characteristics, such as age.
10
Molecular Signaling Pathway Assay Protocol
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Thidiazuron and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reagent for the MTT assay were attained from Sigma‐Aldrich (St. Louis, MO, USA). The penicillin‐streptomycin‐neomycin, and Dulbecco's‐modified Eagle's F‐12 medium (DMEM/F12) were acquired from GIBCO BRL/Invitrogen. Antibodies specific for MMP9, MMP2, uPAR, uPA, TIMP1, TIMP2, caspase‐3, PAI‐1, NF‐κB (p65), phos‐IKK, VEGF, PARP, p‐AKT, AKT, p‐PI3K, PI3K, lamin‐B1, β‐actin and IKK were bought from Thermo Scientific. F‐actin (Alexa Fluor 488 Phalloidin) was purchased from Thermo Scientific.
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