2‐Methylamino‐1‐(p‐tolyl)propan‐1‐one hydrochloride (mephedrone, MW: 213.70), 2‐amino‐1‐(p‐tolyl)propan‐1‐one hydrochloride (nor‐mephedrone, MW: 199.68) and 1‐(4‐(hydroxymethyl)phenyl)‐2‐(methylamino)propan‐1‐one hydrochloride (4‐OH‐mephedrone, MW: 229.70) were synthesized as racemic mixtures. In the case of 2‐(methylamino)‐1‐(p‐tolyl)propan‐1‐ol hydrochloride (dihydromephedrone, MW: 215.72), all four stereoisomers [syn‐(1R,2R), syn‐(1S,2S), anti‐(1R,2S) and anti‐(1S,2R)] were synthesized in their enantiopure form (99%) and tested as 1:1:1:1 mixture. Synthetic procedures and chemical characterization data are given in detail in the Supporting Information . Reagents used in the experiments for uptake inhibition and release in HEK293 cells were used as mentioned in Hofmaier et al., 2014 . Plasmids encoding human SERT were a generous gift of Dr Randy D. Blakely. For uptake and release experiments in HEK293‐cells and rat brain synaptosomes, [3H]‐1‐methyl‐4‐phenylpyridinium ([3H]‐MPP+; 80–85 μCi mmol−1) and [3H]‐5‐HT (28.3 μCi mmol−1) were purchased from American Radiolabeled Chemicals (St. Louis, MO, USA) and Perkin Elmer (Boston, MA, USA) respectively. All other chemicals and cell culture supplies were from Sigma‐Aldrich (St. Louis, MO, USA) with the exception of cell culture dishes, which were obtained from Sarstedt (Nuembrecht, Germany).
Cell culture dishes
Manufactured by Sarstedt
Sourced in Germany, United States, Spain
Cell culture dishes are sterile, disposable containers designed for the in vitro cultivation of cells. They provide a controlled environment for the growth and maintenance of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
3h 5 ht, by PerkinElmer (3 mentions)
3h mpp, by American Radiolabeled Chemicals (2 mentions)
Fetal bovine serum of south american origin, by Thermo Fisher Scientific (2 mentions)
Trypsin edta solution, by Lonza (2 mentions)
3h 1 methyl 4 phenylpyridinium, by American Radiolabeled Chemicals (1 mentions)
Cell culture supplies, by Merck Group (1 mentions)
3h 1 methyl 4 phenylpyridinium 3h mpp, by American Radiolabeled Chemicals (1 mentions)
Paroxetine, by Santa Cruz Biotechnology (1 mentions)
Gbr12909, by Merck Group (1 mentions)
P chloroamphetamine pca, by Merck Group (1 mentions)
White 96 well plate, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
3h imipramine, by PerkinElmer (1 mentions)
Dna restriction enzymes, by New England Biolabs (1 mentions)
T4 dna ligase enzyme, by New England Biolabs (1 mentions)
Reduced glutathione, by Merck Group (1 mentions)
Silver perchlorate, by Merck Group (1 mentions)
9 protocols using cell culture dishes
1
Synthesis and Characterization of Cathinone Derivatives
2
Cytotoxicity and Oxidative Stress Assays
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3,4,5-Dimethylthiazol-2,5-diphenyl-tetrazolium bromide (MTT), carboxy-2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO) were acquired from Sigma (Madrid, Spain). Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin (5000 U/mL), phosphate buffered saline (PBS) and trypsin/EDTA solution (170,000 U/L) were purchased from Lonza (Madrid, Spain). Fetal bovine serum (FBS) of South American origin (Hyclone, GE Healthcare, Logan, UK) was obtained from Thermo Scientific (Madrid, Spain). Cell culture dishes were obtained from Sarstedt (Barcelona, Spain).
3
Synthesis and Characterization of Fluorinated Morpholines
4
Radioligand Binding Assay for Monoamine Transporters
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D‐Amphetamine (D‐Amph) was sourced from Tocris (UK), paroxetine from Santa Cruz (USA), p‐chloroamphetamine (pCA) and GBR12909 were from Sigma‐Aldrich (USA). Hellmanex was from Hellma France S.A.R.L. (Paris, France). Radiochemicals are [3H] 1‐methyl‐4‐phenylpyridinium ([3H]MPP+; 80–85 μCi × mmol−1) from American Radiolabeled Chemicals (St. Louis, MO, USA) and [3H]5‐HT (28.3 μCi x mmol−1) from PerkinElmer (Boston, MA, USA). Other chemicals and cell culture supplies were obtained from Sigma‐Aldrich (St. Louis, MO, USA), and cell culture dishes were from Sarstedt (Nuembrecht, Germany).
5
Serotonin Transporter Radioligand Binding Assay
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DMEM, fetal bovine serum, trypsin and penicillin–streptomycin was purchased from Life Technologies. DNA restriction enzymes and T4 DNA ligase enzyme were from New England Biolabs. Cell culture dishes were from Sarstedt AG & Co, and white 96-well plates were from Nunc. [3H]5-HT (27–28 Ci mmol−1), [3H]imipramine (41 Ci mmol−1), MicroScint-0 and -20 scintillation fluid and 96-well glass fibre filter plates were obtained from PerkinElmer Life Sciences. [3H]vortioxetine (69 Ci mmol−1) was purchased from Ubichem Pharma Services (Hungary). azF and BzF were from Chem-Impex International, Inc. Rho1D4 mAb was obtained from The National Cell Culture Center.
6
Cell Viability Assay Protocol
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3,4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), reduced glutathione, silver perchlorate, and silver tetrafluoroborate were acquired from Sigma-Aldrich (Madrid, Spain). Dulbecco's Modified Eagle's Medium (DMEM), penicillin/streptomycin (5,000 U/mL), phosphate buffered saline (PBS) and trypsin/EDTA solution (170,000 U/L) were purchased from Lonza (Barcelona, Spain). Fetal bovine serum (FBS) of South American origin (Hyclone, South Logan, UT, USA) was obtained from Thermo Scientific (Waltham, MA USA). Cell culture dishes were obtained from Sarstedt (Nümbrecht, Germany).
7
Clonogenic assay for NSCLC cells
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NSCLC cells were seeded in duplicate in Cell + culture dishes (Sarstedt, Landskrona, Sweden) at a density of 500 cells/100 mm dish and were after 24 h treated with cisplatin (2.5-20 μM, Hospira Nordic AB, Stockholm, Sweden) for one hour. Cells were rinsed in PBS after treatment and allowed to form colonies over a 9-days period. The resulting colonies were visualized by staining with crystal violet (0.5 % crystal violet in 25 % methanol) or collected for RNA extraction (see below). For clonogenic survival analyses, colonies consisting of at least 50 cells were counted under a light microscope using duplicate plates from three independent experiments. For retreatment experiments, cell colonies were instead trypsinized and pooled, counted and seeded in 96-well plates for MTT or in new Cell + plates for treatment the next day using the same setup as in the first treatment.
8
Colony Formation Assay of AML Cells
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Cell lines were seeded in cell culture dishes (35 × 10 mm, Sarstedt AG & Co. KG, Nümbrecht, Germany) at a density of 250 cells/mL in 1 mL of methylcellulose-based semi-solid medium (Methocult H4230, Stemcell Technologies, Vancouver, BC, Canada) supplemented with different concentrations of Ara-C. For shRNA experiments, the colony formation capacity of AML cell lines with GLI3 knockdown were compared to that of the negative controls containing non-targeting shRNA. After 7 days, the number of colonies was counted using an inverted microscope (Axiovert 25, Zeiss, Jena, Germany).
9
HPLC Analysis Setup and Materials
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For HPLC analysis, milli-Q grade water was produced in-house by a Milli-Q® Integral 3 purification system (Merck Millipore, MA, USA). Methanol (HPLC grade) was purchased from Scharlab (Barcelona, Spain) and glacial acetic acid, from Sigma-Aldrich (Madrid, Spain). Demineralized water with electrical conductivity of 7-8 µS/cm was obtained in-house by a reverse osmosis unit (Genius 300, Filtec Depuradoras, Girona, Cell culture dishes were obtained from Sarstedt (Barcelona, Spain).
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