This allows analysis of changes in the thermal stability of cellular AR following compound addition as evidence of target engagement.
In antagonist mode, 1α, 25-Dihydroxy Vitamin D3 (3 nM, final concentration) was added immediately after test compound addition.
For all data described in this paper the cardiac microtissues were of good structural integrity at baseline/prior to compound addition.
The FlexStationII (Molecular Devices) is a plate-reader with liquid dispensing capability, ideal for measuring kinetic responses in live cells following compound addition.
The graph reveals that the effect of Rho inhibition can be assessed as early as 36 h after compound addition (48 h after cell seeding) with a larger confidence at the 72 h time point of growth where there is more than twofold difference in the level of cell viability between the samples with and without BCM addition.
Induction of apoptosis was determined by the cleavage of caspase 3/7 at 8, 16, 24 and 48 h after compound addition.
Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 × 105 cells/mL in 100 μL of medium.
Briefly, 100 mL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 × 105 cells/mL in 100 mL of medium.
Briefly, 100 μL of adherent cells (HeLa, NCI-H460, PC3, KB-3-1, MCF-7) waseededed into each well of a 96-well cell culture plate and allowed to adhere for 24 h before test compound addition, while suspended cells (K562) were seeded just before test compound addition, both with an initial density of 1 × 105 cells/mL in 100 μL of medium.
