Pi3k activation dual detection kit

Efficiency of phosphorylation inhibition of the epidermal growth factor receptor (EGFR) or phosphoinositide 3-kinase (PI3K) pathway was assessed using MUSE^® Cell Analyzer (Merck Millipore, Germany) using a Millipore’s Muse™ Activation Dual Detection kits (Merck Millipore, Germany). HDFa cells were seeded in 6-well plates at a density of 2 × 10⁵ per well. Following an overnight incubation, standard growth medium was substituted with the medium containing 0.05% DMSO only, supplemented with 100 ng/ml EGF in 0.05% DMSO, or with 100 ng/ml EGF in 0.05% DMSO and particular tested compounds at the indicated final concentrations21 (link). After 24 hours EGFR pathway activation was determined by Muse^® EGFR-RTK Activation Dual Detection kit (Merck Millipore, Germany) by using a phospho-specific anti-phospho-EGFR (Tyr1173)-Alexa Fluor^®555 and an anti-EGFR-PECy5 according to the manufacturer’s protocol. Similarly, PI3 kinase pathway activation was assessed using an anti-phospho-Akt (Ser473), Alexa Fluor^®555 and an anti-Akt, PECy5 provided in the PI3K Activation Dual Detection kit (Merck Millipore, Germany). Additionally, Jurkat T cells treated with 0.5 μM wortmannin were used as a control of PI3K activation status determination.