Inhibition of phospho-Tau (Thr217) in endogenous cellular models

MCF7 cells were seeded in a 96-well plate(40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 6 hours with increasing concentrations of Staurosporine prepared in media containing 1% FBS. After treatment, the cells were lysed with 100 µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). Tau Phospho(Thr217) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus™ using standard AlphaLISA settings.

As expected, Staurosporine (a broad-spectrum protein kinase inhibitor) triggered a dose-dependent decrease in the levels of Phospho-Tau (Thr217).

SH-SY5Y cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated for 96 hours at 37°C, 5% CO2. The cells were treated for 6 hours with increasing concentrations of Staurosporine prepared in media containing 1% FBS.

After treatment, the cells were lysed with 100 µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). Tau Phospho(Thr217) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus using standard AlphaLISA settings.