| Assay Method Information | |
| | PIKfyve EEA1 Assay |
| Description: | Genetic or pharmacological disruption of PIKfyve activity results in enlargement of endosomal vesicles. This enlargement was utilized as a surrogate readout of PIKFyve inhibition for routine triage of PIKfyve inhibitors. U2OS cells grown in 96-well assay plates were treated with compound diluted in DMEM media containing 10% fetal bovine serum. After 3 hours of treatment, cells were fixed with paraformaldehyde, permeabilized with 0.2% Triton-X in phosphate buffered saline and stained against EEA1. During the secondary antibody staining, cells were also stained with CellMask™ Deep Red and Hoechst to detect cytoplasms and nuclei respectively. Endosomal structures were visualized using a high content imager at 40× magnification. Images were analyzed using a linear classifier algorithm integrating EEA1 spot size, intensity and texture trained on images of cells treated with the potent reference compound APY0201. Compound activity was calculated by subtracting the DMSO signal and calculating percentage activity relative to maximal APY0201 activity. IC50 values were then calculated from concentration versus percent inhibition data by logistic regression. |
| Affinity data for this assay | |
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