Staining for Endogenous Alkaline Phosphatase Activity

The yield and growth of PGCs can be measured by staining the cultures for endogenous alkaline phosphatase activity. Unfortunately, the cells must be fixed in order to stain for AP, so it is a good idea to have plenty of starting cultures so that a few wells can be stained every day in order to monitor PGC growth.

1. Wash cultures twice in PBS. Washing is done by adding fresh PBS, swirling the plate gently, removing the PBS, and adding the fresh PBS. A long incubation is not necessary.

2. Fix cultures in paraformaldehyde for 20 min at room temperature (see Note 5).

3. Wash twice in PBS.

5. Stain for 15-20 min at room temperature, or until PGCs are a reddish-brown color. This can be monitored in the microscope.

6. Wash twice in PBS, and leave cultures in liquid to photograph.

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