Note

This protocol is specific to OGB-1/AM (Oregon Green) and may vary if you use different dyes.

Prepare your samples by measuring their PCV (or estimating the cell amount according to the doubling rate). Dilute them with PBS in order to have between 200 and 500 cells/µl. Prepare at least one well that has seed cells.

For these experiments, we usually use 200'000 cells.

1. Add 5 µl 80% DMSO, 20% pluronic acid to 50 g tube of OGB-1/AM to dissolve dye powder. Mix with 45 µl of PBS for a 1 mM solution. (This is for 10 samples.)

2. Add 5 µl 1 mM OGB-1/AM to 495 µl PBS (for one sample).

3. Add 500 µl directly to the cell culture medium and incubate for 45 minutes at 37 degrees.

4. Centrifuge cells and aspirate culture media and OGB-1/AM. Resuspend in 750 µl of PBS.

5. Centrifuge cells and aspirate PBS. Resuspend in PBS. Repeat this step twice.

6. Aspirate the PBS and add fresh culture medium to the cells.

7. Your cells are ready to go under the scope! Put them on a slide or in an optical well plate quickly to minimize the number of dead cells you observe. If the pathway you use is photosensitive, remember to keep the cells in the dark.