WangLab:EGF Experiment

Time for experiment

 * 30-60 minutes

EGF Experiment

 * 1) Set the cell culture dish on the stage of microscope.
 * 2) *Change the dish cover to a piece of flat cover glass.
 * 3) *Make sure the dish is securely settled on the rack of the stage.
 * 4) *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.
 * 2) *Enable background subtraction of a constant
 * 3) *The constants are chosen by image histograms.
 * 4) *Define regions for tracing change of FRET.
 * 5) Aquire 5 images at 60 seconds intervals.


 * 1) Pause aquiring.
 * 2) *Adding 20 ul x 20 ug/ml EGF to 2 ml dish (200ng/ml), to the edge of dish.
 * 3) *Pipet (big, 1000ul) up and down 3 times to mix well.
 * 4) *Be careful not to touch anything or cause the cell culture dish to move.
 * 5) *Add events
 * 6) **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.