TEVA was performed once the volume of the PDX (#7157) had reached ~500 mm3. The PDXs were excised aseptically from the mice and cut into 2 × 2 × 2 mm3 tissue explants. The 2 × 2 × 2 mm3 explants were then incubated with the different drugs for 24 h in 48-well tissue culture plates at 37 °C, 95% relative humidity and 5% CO2, under sterile conditions with suitable controls. The 2 × 2 × 2 mm3 explants incubated only in culture media without any drug served as control. DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 20% FBS (Gibco), 1 mM sodium pyruvate (Biological Industries, Haemek, Israel), 2 mM l -glutamine (Biological Industries), 1% penicillin–streptomycin–amphotericin (Biological Industries), 0.1 mM MEM non-essential amino acids (Biological Industries), 10 mM HEPES (Biological Industries), 1% BIO-MYC (Biological Industries), and 50 µg/mL gentamycin (Gibco) was used as the culture medium. The therapeutic agents for this part of the study were BYL719 (2 µM), GDC0032 (500 nM), and AEW541 (1 µM). KI67, TUNEL, and pMAPK staining were measured and the TEVA score was calculated using the following formula: (0.3 × KI67 staining) + (0.3 × TUNEL staining) + (0.3 × MAPK staining).
Penicillin streptomycin amphotericin
Manufactured by Sartorius
Sourced in Israel
Penicillin–streptomycin–amphotericin is a commonly used antibiotic and antimycotic solution for cell culture applications. It contains a combination of penicillin, streptomycin, and amphotericin B, which provide broad-spectrum protection against bacterial and fungal contamination in cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hepes, by Sartorius (3 mentions)
Sodium pyruvate, by Sartorius (3 mentions)
Mem non essential amino acids, by Sartorius (3 mentions)
Bio myc, by Sartorius (3 mentions)
L glutamine, by Sartorius (3 mentions)
Gentamycin, by Thermo Fisher Scientific (2 mentions)
Dmem culture media, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Trypsin versene, by Thermo Fisher Scientific (1 mentions)
Dmem lg, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Nycoprep 1, by Axis-Shield (1 mentions)
5 protocols using penicillin streptomycin amphotericin
1
Tumor Explant Viability Assay (TEVA) Protocol
2
PDX Culture and Drug Treatment Protocol
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When the PDXs (preferably first generation PDX) reached ~500 mm3, they were excised from the mice. Tumors were then cut into 2 × 2 × 2 mm3 tissue explants and cultured on gelatine sponges in 12-well tissue culture plates for specific time points as indicated in the text. The DMEM culture media (Gibco) containing 20% FBS (Gibco), 1 mM sodium pyruvate (Biological Industries), 2 mM l -glutamine (Biological Industries), 1% penicillin/streptomycin/amphotericin (Biological Industries), 0.1 mM MEM non-essential amino acids (Biological Industries), 10 mM HEPES (Biological Industries), 1% BIO-MYC (Biological Industries) and 50 μg ml−1 gentamicin (Gibco). For drug treatment, the 2 × 2 × 2 mm3 explants were treated with 3 μM BAY-876 for indicated time in 37 °C, 5% CO2.
3
Maintenance of Human Lung Cancer Cell Lines
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Human lung cancer cell lines, CL1-0, CL1-5, A549, and H1299 (gift in kind from Pro. Cheng-Wen Wu of the Institute of Biomedical Sciences, Academia Sinica, Taiwan) were maintained in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin–amphotericin (Biological Industries, Kibbutz Beit-Haemek, Israel). Cells were cultured in a standard humidified incubator at 37 °C in a 5% carbon dioxide atmosphere, and the medium was changed twice each week. Cells were grown until they were 80–90% confluent. All cultures were negative for Mycoplasma infection.
4
Differentiation of Bone Marrow MSCs into IPCs
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All study procedures were approved by the Institutional Review Board (Taipei Veterans General Hospital). Bone marrow tissues were gathered from 20 healthy donors with their informed consent. After washing the bone marrow sample twice with phosphate buffered saline (PBS, PH = 7.2), density gradient centrifugation (NycoPrep 1.077, Axis-Shield, Oslo, Norway) was possessed and BM-MSCs were isolated. Rinse the BM-MSCs twice in low glucose DMEM (LG-DMEM, 5.5 mM glucose, Invitrogen, Carlsbad, CA) and culture them at 37°C with 5% humidified CO2 in expansion medium consisting of L-DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin/Amphotericin (Biological Industries, Haifa, Israel). The culture medium was replaced every 3 days and the nonadherent cells were removed. When the adherent BM-MSCs were 90–95% confluent (10–15 days), they were subcultured by Trypsin-Versene (Invitrogen). When the third passage BM-MSC reached 80% confluence, it was provided to differentiate into IPCs by culturing in serum-free high glucose DMEM (HG-DMEM, 25 mM glucose) supplemented with 1% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO). 3 days after, the culture medium was replaced with HG-DMEM supplemented with 10% FBS for another 14 days [12 (link)].
5
Tumor Explant Ex Vivo Analysis Protocol
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When the PDXs (preferably first generation PDX) reached ~500 mm3, they were subjected to Tumor ex vivo Analysis (TEVA). After excising out the PDXs aseptically from mice they were cut into 2 × 2 × 2 mm3 tissue explants and cultured in 48 well tissue culture plates for specific time point as indicated in the text. The DMEM culture media (Gibco) contained 20% FBS (Gibco), 1 mM sodium pyruvate (Biological Industries), 2 mM L-glutamine (Biological Industries), 1% penicillin/streptomycin/amphotericin (Biological Industries), 0.1 mM MEM non-essential amino acids (Biological Industries), 10 mM HEPES (Biological Industries), 1% BIO-MYC (Biological Industries) and 50 ug/ml gentamycin (Gibco). For drug treatment, the 2 × 2 × 2 mm3 explants were treated with different therapeutic drugs for 24 h in 48 well tissue culture plates in 37°C, 5% CO2.
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