Human CRC cell lines, including HCT116, HT29, RKO, SW48, Lim1215, and NCI-H508, were purchased from the American Type Culture Collection and cultured in McCoy’s 5A modified media (Invitrogen). SNU-407 was purchased from AddexBio and cultured in RPMI 1640 (Invitrogen). DR5-knockout (KO) HCT116 cells were previously described (26 (link)). DR5-KO RKO cells were generated by CRISPR/Cas9 using a single guide RNA sequence 5’-CGCGGCGACAACGAGCACAA-3’ as described (27 (link)). Cells were authenticated in 2018 by genotyping and analysis of protein expression by western blotting, and routinely checked for Mycoplasma contamination by PCR. All cell lines were maintained at 37°C and 5% CO2 atmosphere. Cell culture media were supplemented with 10% defined FBS (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20–30% density 24 hr before treatment. DMSO (Sigma) stocks of JQ1, I-BET151 (Apexbio), OTX015, I-BET762, 5-FU, and oxaliplatin (Sigma) were prepared and diluted in cell culture media before adding to cells.
Otx015
Manufactured by Merck Group
Sourced in China, United States
OTX015 is a lab equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of OTX015 is to perform specific tasks and procedures required in a laboratory setting. No further details about the intended use or specific capabilities of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
I bet762, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
Mccoy s 5a modified media, by Thermo Fisher Scientific (1 mentions)
I bet151, by Apexbio (1 mentions)
Snu 407, by Addexbio (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Nci h508, by American Type Culture Collection (1 mentions)
Lim1215, by American Type Culture Collection (1 mentions)
Pd098059, by MedChemExpress (1 mentions)
Mtt assay, by Merck Group (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
High capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Aβ1 42, by AnaSpec (1 mentions)
Anhydrous dimethyl sulfoxide dmso, by Merck Group (1 mentions)
Aβ1 42 hilyte 488, by AnaSpec (1 mentions)
4 protocols using otx015
1
CRC Cell Lines and Drug Treatments
2
Cell Viability Assay for Drug Screening
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MTT assay (Sigma) was used to assess cell viability according to previous descriptions [24 ]. Briefly, cells were seeded in 96‐well plates at a density of 1000 cells/well and subjected to the following treatments according to experimental requirements: 100 ng/mL Dox (Sigma), 10 µmol/L PD098059 (MedChemExpress, Shanghai, China), or 10 nmol/L OTX015 (Sigma). Cell viability was assessed at 12 h to 60 h. At 60 h post treatment, 200 µL MTT (5 mg/mL) was added in each plate well and incubated for 4 h. After that, the microplate reader (Molecular Devices, San Jose, CA, USA) was used to measure the absorbance value at 490 nm. All experiments were performed in triplicate.
3
Microglial Phagocytosis Assay
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Reagents for reverse transcription (High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor) and quantitative PCR (Taqman Assays, TaqMan OpenArray Mouse Phagocytosis Panel, and TaqMan Fast Advanced Master Mix), red fluorescent microspheres (FMS), 2.0 μm, Hoechst 33342, CellMask Orange Actin Tracking Stain, CellMask Green Actin Tracking Stain, RPMI were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Opti-MEM was from Gibco, negative siRNA control was from Ambion, Lipofectamine was from Thermo Fisher Scientific, Inc., Aβ1–42 and Aβ1–42 HiLyte 488 were from AnaSpec, Inc. (Fremont, CA, USA). JQ1, GSK12101517, IBET-762, OTX-015, PFI-1, Cytochalasin D were obtained from Sigma-Aldrich (St. Louis, MO, USA). Heat-inactivated foetal bovine serum (FBS), Accutase solution, penicillin, streptomycin, L-glutamine, 3-(4,5-dimethyl-2-tiazolilo)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide, TRI-reagent, DNase I, dithiothreitol (DTT), anhydrous dimethyl sulfoxide (DMSO), lipopolysaccharide from Escherichia coli O55:B5 (toxicity 3,000,000 U/mg), and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4
MTT Assay for Cell Viability
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Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Sigma-Aldrich). Cells were seeded in 96-well plates at a density of 135.000 cells/mL. Twenty-four hours after seeding, either drug (OTX-015, Sigma-Aldrich) or vehicle (DMSO, Sigma-Aldrich) was added to the media. After 48 h, 10 μL of MTT solution 5 mg/mL (Sigma-Aldrich) was added to each well. Two hours later, MTT was dissolved adding SDS 10% 0.01 M HCl and incubated overnight. The next day, absorbance at 570 nm was measured using a microplate reader (SpectraMax i3x Multi-Mode Microplate Reader, Molecular Devices). Experiments were repeated at least three times and viability was normalised to mean of untreated control, set as 100% viability.
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