Ca²⁺ response to NAADP.

NAADP has been shown to induce Ca²⁺ release in a number of cell systems. In our laboratory we investigate the spatio-temporal properties of the NAADP-induced Ca2+ increase in Asterina pectinifera and Astropecten auranciacus oocytes using a cooled CCD camera and confocal laser scanning imaging.

Procedure:
1. Microinject several immature oocytes with OGBD 10 kDa.
2. Prepare acquisition chamber.
3. Get ready the acquisition setup for experiment.
4. Change the needle in injector for NAADP 100 uM and microinject a couple of oocytes.
5. Transfer oocytes in the acquisition chamber and put on the microscope stage.
6. Start uncaging protocol record changes in the cytoplasmic Ca2+ concentration

Important!

• Somewhat caged NAADP is remains reactive for not more than 10-15 minutes after injection, then responsiveness decreases. Therefore:
  1. Try to perform experiment in the interval 5-10 minutes after injection;
  2. Try to keep the same interval from experiment to experiment beetwin injection and uncaging.

Results:
The figure shows Ca2+ response to NAADP uncaging in immature oocytes of Asterina pectinifera (CCD).

The figure shows Ca2+ response to NAADP uncaging in mature Asterina pectinifera oocytes (CCD).

The figure shows Ca2+ response to NAADP uncaging in mature Asterina pectinifera oocytes measured with confocal microscope.