AIM: Determine whether transfected receptor is a ) present on cell surface b) active and c ) leads to cell activation of G-protein pathways. Identify specific cell of interest and develop high throughput assay. Find molecule to inhibit activity.
Method: Cells were transfected with the receptor, selected clonally and treated with fluo-3 to analyze calcium signaling. They were then imaged under a high resolution microscope at 37C. A picture was taken every 5 seconds, stored and analyzed. Click the play button to see the image data as a video.
This video shows how cells pulse free calcium after receiving a single pulse of stimulant. This shows that the receptor is present on the cell surface, is active and can transduce cell signaling cascades.
To analyze this image, each individual frame was analyzed to determine the identity of every cell and the measured the total intensity for EACH cell in the picture. The data for each cell at specific time-point was then plotted along time on a graph.
Interestingly, though the cell looked asynchronous in the graph, they were shown to be synchronized if imaged very rapidly at an early time point (<60 seconds) as the graph below shows.
Lessons from this analysis:
Single Cells Kinetics:
All individuals within a clone are not identical therefore tesing of the cells for drug development must be done with a large population of defined set of tranfected cells.
Assay Kineticts must match Biological signals and assayed within the first 3 minutes.
Physiological conditions are important
These kinetics were only seen at 37C and not at Room temperature.
RESULT: This assay identified the specific CELL of interest that was consistently active. It was later made into a clone and used to screen a small molecule sample collection.
An imaging assay was developed to match these parameters and drug screening performed. A molecule that showed inhibition activity was identified (bottom right).