Chemotaxis assay

Chemotaxis assay
Grow cells to mid log phase 2-7 x 10^6 cells/ml in shaking culture
Spin down cells
Wash with Na/K buffer
Resuspend in 10ml Na/K buffer
Shake

Pulsing
2-7 x 10^7 cells in 10ml Na/K P buffer
Flowrate: 3.5
Pulse time: 2s (if vol. of cells diff, change this)
Stop time: 358s
Pulse with 12.5ul 30mM cAMP in 50ml Na/K P buffer (30nM cAMP) for 5h

Use 75ul cells in 3ml Na/K buffer in glass dish
Wait 15-20min for attachment onto dish

Turn on microscope
Adjust settings for fluorescent light: Put in polariser, sth else
Add 10ul of 30mM cAMP to needle
Place needle near bottom of dish, almost touching cells
Use 20X lens to find cells and needle
Use 40x to be more precise
Turn on confocal
Start program
Click sequence to start taking pictures
Take picture every 6s for 30min