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Spectramax m5e

Manufactured by Thermo Fisher Scientific

The SpectraMax® M5e is a multi-mode microplate reader from Thermo Fisher Scientific. It is designed for absorbance, fluorescence, and luminescence measurements. The device can perform these measurements in a variety of microplate formats.

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3 protocols using spectramax m5e

1

Evaluating Leukemia and Solid Tumor Cell Lines

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Murine leukemia cell lines were isolated from mice that had developed leukemias carrying different chimeric FGFR1 genes as described previously [18 (link)] and their immunophenotype was confirmed using standard flow cytometry analysis. The KG1 cell line was shown to carry the FGFR1OP2-FGFR1 rearrangement demonstrating the identity of this cell line. Human lung H1581 and H2228 and breast MDA-MB-134VI and T47D cancer cells were purchased from ATCC (passage number < 15). All cell lines were cultured in RPMI (Invitrogen) with 5% FBS (Hyclone), at 37°C in 10% CO2. For drug treatments, 40,000 leukemia cells/well or 5,000 solid tumor cells/well were seeded in 96-well plates and incubated overnight, then treated with the either DMSO (control) or the drugs indicated in the results section at concentrations defined by the experiments. Cell viability was determined using Cell Titer-Glo luminescence cell viability kits (Promega) and a SpectraMax® M5e (Molecular Probe) luminescence plate reader [18 (link)].
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2

Xenograft Model of FGFR Inhibition

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NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (obtained from the Jackson Laboratories), were maintained as a breeding colony. All experiments were conducted under Augusta University IACUC approved protocols. Female, 6–8 week old mice were used in all xenograft experiments. Mice were engrafted with 1–2 × 106 cells via tail vein injection. All mice were treated with either drug, or vehicle control (PEG300:acetic buffer = 1:1), orally using a gavage needle once per day. All treatments were performed 5 days per week for 4 weeks.
Ponatinib was provided by Ariad Pharmaceuticals Inc. (Cambridge, MA). PD173074 was purchased from Cayman Chemical, AZD4547 and BGJ398 from ChemieTek, JNJ-42756493 from Active Biochem and TKI258 from LC Laboratories. E3810 was provided by EOS pharmaceuticals. All drugs were dissolved in DMSO and stored at −80°C before use. For drug treatments, cells were seeded at 3,000–10,000 cells/well, depending on the cell line, in 96-well plates and incubated overnight. Cells were then treated with either DMSO (control) or different FGFR inhibitors as indicated in the results section at concentrations defined by the experiments. Cell viability was determined using CellTiter-Glo® luminescence cell viability kits (Promega) and a SpectraMax® M5e (Molecular Probes) luminescence plate reader as described previously.8 (link)
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3

Quantifying ROS in ARPE-19 Cells

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Commercially available dye, CMH2DCFDA (Invitrogen) was used to measure the production of reactive oxygen species (ROS) in ARPE-19 cells. These cells were grown overnight in a 96 well plate with exposure to room-air or CSE (10%) and treated with cysteamine (250 μM, Sigma) or fisetin (40 μM, Sigma) for 8 hours. Cells were then incubated with 10μM of CMH2DCFDA for 30 minutes. Changes in fluorescence were quantified using excitation (495 nm) and emission (515 nm) wavelengths recorded on SpectraMax M5e (Molecular Probes) plate reader, and the SoftMax Pro 6.0 analysis software as described recently [24 (link)–26 (link)].
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