Flou-4AM MW =1000 dissolve in DMSO to 1-5mM; use 2.5uM link

Protocol

1. 24 hours before, change media
2. 30min before acquistion start heating confocal
3. 30 min before acquisition, load with 2.5uM or (5uM) Fluo-4 (search for molarity), I use normal RPMI + B27 but some without phenol red would be even better
4. 15min before acquisition move to 37C confocal, Heating on, CO2 on
5. Find the beating clusters/cells positions using BF and save positions
6. For imaging set linescan 500 per sec 256x256 px binned2x, pinhole 100, laser power 2-8,
7. in BF find the spot
8. put the arrow in the middle
9. do the best focus with BF
10. switch to LIVE, linescanning
11. linescan
12. if it looks okey then do 15 sec at 500fps