Equipment and chemicals
Zeiss inverted microscope, eppendolf
microinjector, eppendolf micromanipulator, (in Dr.
Eppendolf femtotips, 100mM cAMP in PM,
DB buffer, PM Buffer.
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
Carefully position the femtotip (filled with 100
mM cAMP) to
the center of the field .
Videotape the process for 5-10 min.