PAB was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) with a purity of >98%. PAB was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution, which was then diluted using McCoy's 5A (modified) medium (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) to a final concentration of DMSO of <0.05%, which was considered to have no detectable effects on cell growth and viability. Trypsin, EDTA, PBS, monodansylcadaverine (MDC), propidium iodide (PI) and Annexin V were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). McCoy's 5A (modified) medium and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Primary antibodies directed against microtubule-associated proteins 1/2 light chain 3 (LC3-I/II), autophagy protein 5 (ATG5), β-actin, phosphorylated (p)-Ras, p-RAF proto-oncogene serine/threonine-protein kinase (Raf) and extracellular signal-regulated kinase (ERK) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were also obtained from Cell Signaling Technology, Inc. All other chemical reagents used in the study were of analytical reagent grade.
Mccoy s 5a modified medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, Canada, Belgium
McCoy's 5A modified medium is a cell culture medium formulated for the growth and maintenance of various cell types. It provides the necessary nutrients and components to support cell proliferation and viability in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (123 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (42 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (38 mentions)
Streptomycin, by Thermo Fisher Scientific (36 mentions)
Penicillin, by Thermo Fisher Scientific (35 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (32 mentions)
Hct116, by American Type Culture Collection (20 mentions)
Glutamax, by Thermo Fisher Scientific (18 mentions)
Hct116, by Thermo Fisher Scientific (17 mentions)
Rpmi 1640, by Thermo Fisher Scientific (12 mentions)
L glutamine, by Thermo Fisher Scientific (11 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (11 mentions)
Dmem f12, by Thermo Fisher Scientific (11 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (10 mentions)
Sw480, by American Type Culture Collection (9 mentions)
Hydrocortisone, by Merck Group (9 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (9 mentions)
Dmem medium, by Thermo Fisher Scientific (8 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (8 mentions)
Cholera toxin, by Merck Group (7 mentions)
258 protocols using mccoy s 5a modified medium
1
Autophagy Induction by PAB in Cancer Cells
2
Osteosarcoma Cell Line Culturing and Flavopiridol
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Osteosarcoma cell lines U2OS, SaOS-2, SJSA-1, and 143B were purchased from the American Type Culture Collection (ATCC). Cells were cultured in a humidified atmosphere at 37° C and 5% CO2. U2OS were cultured in McCoy's 5A Modified Medium (Gibco) supplemented with 10% BCS (Sigma) and 1% streptomycin/penicillin (Gibco). SaOS-2 were cultured in McCoy's 5A Modified Medium (Gibco), supplemented with 15% BCS (Sigma) and 1% streptomycin/penicillin (Gibco). SJSA-1 were cultured in RPMI 1640 (Gibco), supplemented with 10% BCS, 1% streptomycin/penicillin (Gibco) and 1% GlutaMAX (Gibco). 143B were cultured in MEM (Gibco), supplemented with 10% BCS, 1% streptomycin/penicillin (Gibco), 0.015 mg/ml BrdU (Sigma), and 1% GlutaMAX (Gibco). Flavopiridol (Alvocidib) was purchased from Selleck Chemicals (Houston, TX, USA).
3
Preparation and Characterization of Delta Inulin
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Delta inulin was prepared and supplied by our industrial partner Vaxine Pty Ltd. (Adelaide, Australia) by a method previously described [32 (link)]. PCA, sodium metaperiodate, sodium acetate, acetic acid, glycerol, ethanolamine, ethylenediamine, acetonitrile, sodium cyanoborohydride solution (5 M in 1 M NaOH), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and phosphate buffered saline tablets (PBS, pH 7.4, 0.01 M), 6-[Fluorescein-5(6) carboxamido] hexanoic acid (FITC) were purchased from Sigma-Aldrich Castle Hill, New South Wales Australia. Dimethyl sulfoxide (DMSO) for cell work were supplied by Merck (New South Wales, Australia). The NMR solvents, deuterated water (D2O) and DMSO (DMSO-D6), were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). All reagents and chemicals used were of analytic grade. General cell culture was supplied as a gift from Vaxine Pty Ltd. For the biological work, cell media and staining reagents Dulbecco’s Modified Eagle Medium (DMEM), l -Glutamine, trypsin and McCoy’s 5A (Modified) Medium were all purchased from Thermo Fisher Scientific (Thebarton, Adelaide, Australia). High purity (Milli-Q) water obtained from Sartorius™ Arium Pro-Ultrapure Water System was used in all of the experiments.
4
Culturing Human Cancer Cell Lines
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PC-3, DU-145 (human prostate cancer) and SKOV-3 (human ovarian cancer) cell lines were grown in RPMI-1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and McCoy’s 5a Modified Medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated foetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA), respectively. Cells were cultured at 37 °C, 5% CO2 in a humidified incubator. Cells were tested regularly for mycoplasma contamination.
5
Culturing Human Breast Cancer Cell Lines
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Human breast cancer cell line MDA-MB-231 was cultured in Leibovitz's L-15 (Thermo Fisher Scientific Inc. United States. Gibco. 11415064) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc. United States. Gibco. 10099141) and human breast cancer cell line SK-BR-3 was cultured in McCoy's 5a modified medium (Thermo Fisher Scientific Inc. United States. 16600082) containing 10% FBS. All the cells were incubated in a humidified incubator containing 5% CO2 at 37°C.
6
Colon Cancer Cell Line Culture Protocols
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The SW480, DLD1, COLO201, HCT116, and LS174T colon cancer cell lines were obtained from the American Type Culture Collection. SW480, DLD1, and COLO201 cells were grown in RPMI-1640 growth medium (Thermo Fisher; 11875093), HCT116 cells were grown in McCoy's 5A (Modified) medium (Thermo Fisher; 16600082), and LS174T cells were grown in Minimum Essential medium (Thermo Fisher; 11095080). All growth media were supplemented to 10% FBS (Thermo Fisher; 10082147) and 2mM L-glutamine (Thermo Fisher; 25030149). Cells were incubated at 37°C in 5% CO2. No antibiotics were used. Proper cell line identity was confirmed using STR profiling and the absence of mycoplasma contamination was confirmed routinely using PCR (Genlantis; MY01050).
7
Comparison of Breast Cell Lines
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Different cell lines (all from American Type Culture Collection ATCC, Manassas, VA, USA) were used to compare the results of primarily isolated cells to established models. The mammary epithelial cell line MCF-10A (ATCC® CRL-10317™) was cultivated in Mammary Epithelial Cell Growth Medium (PromoCell GmbH, Heidelberg, Germany) with the addition of 100 ng/ml cholera toxin (Sigma Aldrich). Furthermore, we used six different breast cancer cell lines. BT-474 (ATCC® HTB-20™, luminal B) were cultivated in Hybri-Care Medium (ATCC), 10% FBS, 1.5 g/l NaHCO3 (Sigma Aldrich). MCF-7 (ATCC® HTB-22™, HR-positive) were cultivated in DMEM, 10% FBS, 2 mM L-glutamine. MDA-MB-231 (ATCC® HTB-26™, TNBC, mesenchymal-like) were cultivated in DMEM, 10% FBS, 2 mM L-glutamine. SKBR3 (ATCC® HTB-30™, HER2-positive) were cultivated in McCoy’s 5a Modified Medium (Thermo Fisher Scientific Inc.), 10% FBS. MDA-MB-468 (ATCC® HTB-132™, TNBC, basal-like) were cultivated in DMEM/F12 Medium (Biochrom), 10% FBS. T-47D (ATCC® HTB-133™, Luminal A) were cultivated in RPMI-1640 (Thermo Fisher Scientific Inc.), 10% FBS.
8
Rat Mammary Adenocarcinoma Tumor Resection
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All animal procedures were conducted in accordance with protocols approved by the Illinois Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign. All experiments in this study were carried out in compliance with the ARRIVE guidelines. Approximately 9 × 106 MAT B III rat mammary adenocarcinoma cells (CRL-1666, American Type Culture Collection) were grown in the same media comprised of McCoy’s 5A (Modified) medium (16600082, Thermo Fisher Scientific) supplemented with 10% v/v fetal bovine serum (16140071, Thermo Fisher Scientific, Waltham, MA, USA) and 1% v/v Penicillin–Streptomycin (10378016, Thermo Fisher Scientific), and were injected subcutaneously into a healthy rat. After 7 days, when the tumor was approximately 5 × 5 × 2 mm3, the rat was euthanized by isoflurane overdose. Approximately 15 min postmortem, the tumor was surgically resected and placed in an imaging dish containing cold saline.
9
Culturing and Validating Human Cell Lines
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Human cell line U2OS cells (ATCC, #HTB-96) used for experimental validation of the model were cultured in McCoy’s 5A Modified Medium (Thermo Fisher Scientific, #16600–082) supplemented with 10% Fetal bovine serum (FBS) (Thermo Fisher Scientific, #16000–044) and 100 μg/ml penicillin-streptomycin antibiotics (Thermo Fisher Scientific, #15140–122). Human cell line A549 cells (ATCC, #CCL-185) used for experimental validation of the model were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, #11965092) supplemented with 10% Fetal bovine serum (FBS) (Thermo Fisher Scientific, #16000–044) and 100 μg/ml penicillin-streptomycin antibiotics (Thermo Fisher Scientific, #15140–122). Experiments were performed on cells from passages 5–10. All cells were maintained under recommended conditions at 37°C and 5% CO2. Cells were verified to have no detectable mycoplasma contamination (ATCC, #30–1012K) prior to starting experiments.
10
Engineered HCT 116-19 Cell Line
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HCT 116 cells were acquired from ATCC (American Type Cell Culture, Manassas, VA). The HCT 116–19 was created by integrating a pEGFP-N3 vector (Clontech, Palo Alto, CA) containing a mutated eGFP gene. The mutated eGFP gene has a nonsense mutation at position +67 resulting in a nonfunctional eGFP protein. For these experiments, HCT 116–19 cells were cultured in McCoy’s 5A Modified medium (Thermo Scientific, Pittsburgh, PA) supplemented with 10% fetal bovine serum, 2mM L-Glutamine, and 1% Penicillin/Streptomycin. Cells were maintained at 37°C and 5% CO2. The eGFP targeting custom designed 72-mer oligonucleotide was synthesized by IDT (Integrated DNA Technologies, Coralville, IA).
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