Migration assays were performed as previously described54 . Freshly isolated BMDMs (100,000 cells) were seeded in the top chamber of a 24-well PET membrane (8 mm pore size). Cells translocated to the lower chamber in response to exposure in the lower chamber of vehicle or 100 ng/ml CX3CL1 (472-FF; R&D) in the presence or absence of either an AKT inhibitor (MK-2206; 10 μM, Cayman), PI3K inhibitor (BEZ235; 10 μM, Cayman), EGFR inhibitor (erlotinib, 10 μM, LC laboratories) or NF-κB inhibitor (JSH23, 30 μM, Sigma-Aldrich) for 3 h. Cells in the upper chamber were removed with a cotton swab, and the filters were fixed with 70% ethanol and stained with 2% crystal violet. Filters were photographed on a Leica DMi1 microscope and total cell number was counted.
Bez235
Manufactured by Cayman Chemical
Sourced in United States
BEZ235 is a synthetic organic compound used as a laboratory reagent. It is a mTOR inhibitor that can be utilized in various research applications. Further details on its specific functions or intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Mk 2206, by Cayman Chemical (2 mentions)
Jsh 23, by Merck Group (1 mentions)
Dmi1 microscope, by Leica (1 mentions)
Erlotinib, by LC Laboratories (1 mentions)
Protein lysis buffer, by Merck Group (1 mentions)
Triton 100, by Merck Group (1 mentions)
Tris buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit antihuman α tubulin antibody, by Cell Signaling Technology (1 mentions)
Alcl3 6h2o, by Merck Group (1 mentions)
Mgcl2 6h2o, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Perifosine, by Merck Group (1 mentions)
Erlotinib, by Selleck Chemicals (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Puromycin, by Lonza (1 mentions)
Wortmannin, by Selleck Chemicals (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
6 protocols using bez235
1
Chemotaxis Assay for Macrophage Migration
2
Apoptosis Assay for Cell Viability
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MgCl2 ·6H2O, AlCl3·6H2O, NaOH, 5-FU, Tween® 20, protein lysis buffer, and Triton® 100 were purchased from Sigma-Aldrich Corp (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), PBS buffer, and Tris buffer were obtained from Life Technologies Corporation (Mulgrave, Victoria, Australia). BEZ-235 was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). The Annexin V-FITC Apoptosis detection kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Mouse monoclonal antihuman Bcl-2 antibody and rabbit antihuman α-tubulin antibody were obtained from Cell Signaling Technology Inc (Danvers, MA, USA).
3
Drug Combination Therapy Effects on Tumor Spheres
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Canertinib (CI-1033, Pfizer Pharmaceuticals) and Erlotinib (Selleck Chemicals) were kindly provided by Dr. Mukesh Nyati at University of Michigan. Perifosine (Sigma), MK2206 (Selleck Chemicals) and BEZ235 (Cayman Chemical) were kindly provided by Dr. Alnawaz Rehemtulla at University of Michigan. For drug treatment, we used CI-1033 at 2 μM; Erlotinib at 3 μM, 6 μM and 10 μM, MK2206 at 0.25 μM, 0.5 μM and 1 μM; Perifosine at 1 μM, 3 μM and 5 μM; and BEZ235 at 25 nM, 125 nM and 250 nM. Above drugs were used to treat H1299 cells in tumor sphere formation assay for 4 days. Individualized drug concentrations were set according to cellular response to each drug for a common period of time.
4
Cell Culture Media and Supplements
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Cell culture media and supplements such as RPMI 1640, DMEM, PBS, and antibiotics (penicillin, streptomycin, and puromycin) were purchased from Lonza (Allendale, NJ), Fisher Scientific (Pittsburgh, PA) or Santa Cruz Biotechnology (Dallas, TX). Heat-inactivated fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Flowery Branch, GA). Sodium butyrate and sanguinarine were procured from Sigma-Aldrich (St. Louis, MO). Trichostatin A (TSA), apicidin, HC toxin, LY294002, PX866, CAL-101, MK2206, Triciribine, GDC0068, Rapamycin, AZD8055, and BEZ235 were obtained from Cayman Chemical (Ann Arbor, MI). Tetrandrine was acquired from Santa Cruz Biotechnology, and (–)-depudecin was purchased from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA). Datiscetin was ordered from BOC Sciences (Shirley, NY), while wortmannin and CUDC-907 were procured from Selleck Chemicals (Houston, TX).
5
EGF and Growth Factor Treatments
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Human Epidermal growth factor (EGF) was purchased from Upstate Biotechnology (Waltham, MA, USA), FR180204 from Sigma (St.-Louis, MO, USA), Bez235 from Cayman Chemicals (Ann Arbor, MI, USA), and IFNγ, IGF1, and IGF2 from ReliaTech (Wolfenbüttel, Germany). Regular (short acting) insulin was obtained from Berlin Chemie (Berlin, Germany).
6
K562 Cell Culture with Doxo and BEZ235
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K562 and K562/A cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO2. Doxo (cat. no. 22386; Cayman Chemical Company) and BEZ235 (cat. no. 21185; Cayman Chemical Company) were diluted in DMSO (Merck KGaA) at a concentration of 1 mM as the primary stock solution and stored at 4°C, respectively.
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