note: this protocol is specific to OGB-1/AM (oregon green) and may vary if you used 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 would like 200,000 cells in a volume

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

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

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

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

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

6.Aspirate off the PBS and Add fresh culture media 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.