CellSensor™ irf1-bla TF-1 Cell Line

Jak/Stat signaling pathways play essential roles in cellular responses to distinct cytokines. One of the Jak/Stat pathways, Jak2/Stat5, mediates cell proliferation in response to Interleukin-3 (IL-3), prolactin, erythropoietin (Epo), and granulocyte-macrophage colony stimulating factor (GM-CSF). JAK2 gene knockout causes embryonic lethality due to defective erythropoiesis, suggesting that the Jak2/Stat5 pathway plays an important role in red blood cell formation.

The recent discovery of an activating mutation in JAK2 (V617F) present in a high percentage of myeloproliferative disease (MPD) patients suggests that the Jak2/Stat5 pathway may be a potential therapeutic target for certain forms of MPD. The activated Stat5 transcription factor recognizes and binds to a specific palindromic DNA sequence found in the promoter region of β-casein, interferon regulatory factor-1 (irf-1), and a number of other genes. The CellSensor™ irf1-bla TF1 Cell Line contains a beta-lactamase reporter gene under control of the irf-1 response element stably integrated into TF1 cells. TF1 cells are a human erythroleukemia cell line that is growth-dependent on GM-CSF and have an intact GM-CSF-JAK2-STAT5 pathway.

This cell line is validated for EC₅₀ and Z'-factor using GM-CSF. This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to EPO and Jak Inhibitor 1 was also tested (Figures 2 and 3). CellSensor™ irf1-bla TF1 cells were stimulated in triplicate with GM-CSF over the indicated concentration range in a 384-well format. Cells were incubated for 5 hr. with agonist and 0.5% DMSO and then combined with LiveBLAzer™-FRET B/G Substrate (CCF4-AM) for 2.5 hr. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The 460/530 ratios were plotted for each replicate against the indicated concentrations of GM-CSF. CellSensor™ irf1-bla TF1 cells were stimulated with Epo over the indicated concentration range in a 384-well format.

The Response Ratios were plotted against the indicated concentrations of Epo. CellSensor™ irf1-bla TF1 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384-well format. Cells were then stimulated with GM-CSF or EPO for 5 hr. in 0.5% DMSO and then combined with LiveBLAzer™-FRET B/G Substrate (CCF4 - AM) for 2.5 hr. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. These values were converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 of GM-CSF or EPO-treated cells) and plotted against the indicated concentrations of Jak Inhibitor 1.

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