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        • Stable Cell Lines for Drug Screening in CNS

Gamma-Secretase Activity Assay Cell Line

Reference: P30702


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Data Sheet

Alzheimer’s Disease (AD) is characterized by brain depositions of the beta amyloid (bA). The bA is the amyloid precursor protein (APP) digestion product, which is released from the cell after beta-secretase and  gamma-secretase proteolysis. A novel recombinant cell line has been developed to screen inhibitors of γ-secretase activity via APP processing pathway.

APP-C99 U2OS cell line developped by Innoprot allows to perform assays to evaluate the endogenous γ-secretase proteolityc process in living cells. The 99-amino-acid long transmembrane APP-C99 tagged with tGFP construct is processed by γ-secretase showing a cytoplasmic fluorescence pattern. When γ-secretase is inhibited, fluorescence aggregates appear.


Assay Details


This cell line has been validated using different inhibitory compounds for γ-secretase (positive controls) and ß-secretase activity (negative controls) in an automatic epifluorescent imaging system to acquire and analyze how robust the model is. After treatments, images obtained were analyzed using Attovision software from Becton Dickinson and fluorescent vesicles into the citoplasm were quantified.

IC50 value for DAPT was determined by treating U2OS APP-C99-tGFP cells with inhibitor concentrations from 100pM to 100 µM during 24 hours. Aggregates formation was quantified with a BD Pathway 855 High-Content Bioimager and Attovision software. IC50 for DAPT is 1.10x10e-7 M and Z’ for this experiment was 0,79 +/-0,02.


Applications


  • This model permits evaluate a lybrary of compounds, candidates to inhibitors, in living cells studying the vesicles retention
  • This model provide a strategy to evaluate drug against gamma secretases activity without the necessity to be permeable.
  • This model allows to analyse in the space and time the compund effect in a multiparametric manner.

 

  

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