Kinase Akt Assay Overview

Express a stable, genetically-encoded kinase activity reporter using Incucyte^® Kinase Akt Lentivirus in a variety of cell types to monitor Akt kinase activity in live cells over time. 

Figure 1. Measure compound effects on Akt activity in live cells.  SK-OV-3 cells stably expressing the Incucyte^® Kinase Akt Green/Red indicator were treated with Akt selective inhibitor MK2206, resulting in translocation of the green fluorescent sensor from the cytoplasm to the nucleus.  The kinetic graph on the left shows a decrease in the Nuclear Translocation Ratio over time, indicating Akt inhibition.  The image panel shows the phase and red fluorescence image channels on the top and the green fluorescence channel on the bottom.  Movement of the green fluorescent sensor from the cytoplasm to the nucleus can be seen over 28 minutes, while localization of the red fluorescent nuclear marker does not change. 

Enhance productivity by evaluating multiple compounds or treatments in a 96-well format.  Bridge the gap between single target-focused biochemical assays and lower-throughput cell-based follow-up experiments for the identification of novel treatments.   

Figure 2. Use live-cell kinetic data to evaluate time-dependent effects on Akt activity of multiple compounds from a single 96-well plate.  A549 cells stably expressing the Incucyte^® Kinase Akt Green/Red indicator were seeded in a 96-well plate at 5K cells/well.  Cells were treated with inhibitors targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103.  The 96-well Microplate Graph (left) shows time- and concentration-dependent decreases in the Nuclear Translocation Ratio (NTR) for all compounds tested, indicating Akt inhibition. Kinetic graphs of the NTR over 24 h for AZD5363 and LY294002 (middle) show varying kinetic profiles.  Addition of AZD5363 caused sustained inhibition of Akt over 24 h, while PI3K inhibitor LY294002 caused a transient decrease in NTR followed by a recovery to baseline, indicating reactivation of Akt.  IC50 curves (right) depict 1.5 h and 24 h time point data. 

Gain new insights with sensitive detection of both short term and long term dynamic changes in Akt activity in live cells continuously over time. Automatically measure and visualize inhibition of Akt and utilize kinetic measurements from any time point.  

Figure 3. Observe effects of serum starvation and Akt activation over time.  HeLa cells stably expressing the Incucyte^® Kinase Akt Green/Red indicator were shifted into media containing no serum to remove various growth factors which are known to activate the PI3K/Akt kinase pathway.  The serum shift caused a decrease in the Nuclear Translocation Ratio (NTR), indicating a decrease in Akt activity, while no change was observed in control wells kept in 10% serum.  After 4 h, cells were treated with either 1.1 ng/mL epidermal growth factor (EGF) or 3.3 nM R3-IGF-1 (a recombinant analog of insulin-like growth factor).  Both compounds induced activation of Akt, as demonstrated by a rapid increase in the NTR.  R3-IGF-1 addition resulted in sustained activation over the 12-hour time course, while activation by EGF diminished over time. Representative images from all conditions are shown.  Prior to compound addition, the sensor is localized to the nucleus of cells in serum free media and the cytoplasm of control cells in 10% serum (3 h).  Upon activation by EGF or R3-IGF-1, the sensor translocates from the nucleus to the cytoplasm (4.5 h).   The sensor returns to the nucleus as Akt activity decreases over time in EGF-treated cells, while it remains in the cytoplasm of R3-IGF-1 treated cells (12 h). 

Quantify Akt kinase activity and proliferation within the same assay. Gain insights into the effect of Akt inhibition on cancer cell proliferation, all within the same cell population. 

Figure 4. Make concurrent measurements of Akt activity and cell proliferation within the same assay. MDA-MB-231 and T-47D cells expressing the Incucyte^® Kinase Akt Green/Red indicator were seeded in 96-well plates at 2.5K cells/well and 4K cells/well, respectively. Nuclear Translocation Ratio (NTR) as a measure of Akt activity and Red Object Counts as a measure of cell proliferation were monitored concurrently in the same cells following treatment with a selective Akt inhibitor (MK2206), and a protein synthesis inhibitor (Cycloheximide). Kinetic graphs of NTR (top row) demonstrate concentration-dependent inhibition of Akt by MK2206 in both cell lines. Measurements of Red Object Count from the same cells (middle row) reveal differential effects of Akt inhibition by MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition in T-47D cells but little effect in MDA-MB-231 cells. Cycloheximide treatment caused inhibition of cell proliferation without affecting Akt activity in both cell lines.  IC₅₀ graphs of Red Object Count (24 h) and Nuclear Translocation Ratio (72 h) are shown (bottom row).  The IC₅₀ for Akt inhibition as measured by the NTR was similar in both cell types, while the IC50 for Red Object Count is significantly left-shifted in T-47D cells compared to MDA-MB-231 cells.