Sperm motility

The NP-EGTA-AM was diluted in DMSO at 4 mM stock solutions. We recommend using water-free ampullas of DMSO. 

The incubation protocol was:

- 94 µl sample (in ES solution)

- 5 µl Pluronic 10% (0.05% Final concentration)

- 1 µl NP-EGTA AM Caged Calcium 4 mM (40 µM Final concentration)

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Incubation was performed at room temperature (about 22°C in our lab) for 45 - 60 minutes. 60 minutes incubations sometimes resulted in better responses. We diluted the incubation 1:20 in activation solution. At this point, you have to hurry, as cells are only motile for a short period of time. If cells are sticking to the walls of the observation chamber, you can add 0.05% final pluronic in your final sperm suspension. 

NP-EGTA is a poor caged compound. Therefore, use as much light for uncaging as possible.