One day before transfection, plate 293 cells in growth medium (with 10% fbs) in one T75 flask




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Cell preparation:


Transfection reagent preparation:

Transfection:



  • Wash 293 cells twice with Opti-MEM medium without antibiotics

  • Add DNA-lipofectamine 2000 complexes to the 293 monolayer

  • Incubate at 37C incubator for 5 h

  • Remove DNA-lipofectamine 2000 complexes

  • Wash cells with PBS + 2 mM EDTA once

  • Resuspend the cells in 293 SFM medium with antibiotics (single cell suspension)

  • Add 25 l cell suspension/well into 384-well compound plates (black with clear bottom) (1,000 cells/well)

  • The compound plates contain 10 l/well compound solution prior to the addition of cell suspension. The final concentration for compound carriers, methanol or DMSO, are 0.5% and 0.1%, respectively.

  • After overnight incubation, the AChE activity was measured using Ellman’s assay.


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