WangLab:EGF Experiment

Time for experiment

• 30-60 minutes

EGF Experiment

1. Set the cell culture dish on the stage of microscope.
   • Change the dish cover to a piece of flat cover glass.
   • Make sure the dish is securely settled on the rack of the stage.
   • Make sure the 40x objective touches the bottom of dish.

1. Start Metafluor

1. Open a CFP-YFP-FRET protocal

1. Use FRET-open to locate a good cell.

1. Starting focusing to adjust focusing and exposure time.

1. Configure-> Aquisition to set exposure time.

1. Aquire one image, define regions.
   • Enable background subtraction of a constant
   • The constants are chosen by image histograms.
   • Define regions for tracing change of FRET.
2. Aquire 5 images at 60 seconds intervals.

1. Pause aquiring.
   • Adding 20 ul x 20 ug/ml EGF to 2 ml dish (200ng/ml), to the edge of dish.
   • Pipet (big, 1000ul) up and down 3 times to mix well.
   • Be careful not to touch anything or cause the cell culture dish to move.
   • Add events
     • Events -> EGF -> mark events.

1. Start Aquiring at 30 seconds intervals for 20 cycles and the at 60 seconds intervals for 20 cycles.

1. Adjust Focus if necessary.

Note

• The same protocol can be used for pervanadate experiments by changing EGF to pervanadate.