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b-Galactosidase Color Assay for Cultured Cells 1. For transient expression assays, wait until 2 days after the transfection to begin the color assay. Aspirate the media and rinse the cells twice with PBS. 2. To fix the cells, add 5 ml of a 0.5% Glutaraldehyde in PBS (pH 7.2) solution; incubate @ room temp. for 10 minutes. 3. Rinse the cells twice with PBS. 4. Stain the cells in [5 mM Potassium Ferricyanide, 5 mM Potassium Ferrocyanide, 2 mM MgCl2, and 1 mg/ml X-gal] overnight in the dark @ room temp. Notes: The 25% Glutaraldehyde stock solution is stored @ -20o C. 200 mM stocks of the potassium solutions are prepared in PBS and stored in the dark @ 4o C. 0.01% NaDOC and 0.02% NP-40 may be included in the staining solution, but they are not essential. From the Laboratory of Dr. Allan Bradley Baylor College of Medicine, Houston, Texas