Equipment and chemicals
Zeiss inverted microscope, eppendolf microinjector, eppendolf micromanipulator, (in Dr. Murphy’s lab)
Eppendolf femtotips, 100mM cAMP in PM,
DB buffer, PM Buffer.
Preparing Cells
a. Wash cells with DB and resuspend them to 2 X 107 / ml
b. Shake cells at 120 rpm for 1 hour
c. Pulse cells with 100 nM cAMP for 4 hours
d. Wash cells with PM once and vortex to disperse cells
e. Dilute cells to 2 X 105 / ml and spot 20 microliter on the chamber, wait for 10 min and then fill chamber with PM buffer
Chemotaxis Assay
Carefully position the femtotip (filled with 100 mM cAMP) to the center of the field .
Videotape the process for 5-10 min.
TransWell Chemotaxi