BETA-GALACTOSIDASE (X-GAL) STAINING ASSAY

Adapted from BV’s Cookbook by TCH 8/18/01

To stain T-25 flasks:

1. Remove media from cells. (OPTIONAL: wash the cells with PBS).

2. Add 3ml fixative per T25 and incubate at room temp for 5 min.

3. Remove fixative. (OPTIONAL: rinse with PBS x 2, 2 ml each).

4. (Optional: Add 1ml X-gal stain and remove).

5. Add 2 ml X-gal stain, place cells at 37°C for 30 min to several hours.

6. Cells expressing b-gal will turn blue. Remove green filter from microscope to visualize.

To stain 24 well plates:

1. Remove media from cells. (OPTIONAL: wash the cells with PBS).

2. Add 1.0 ml fixative/well and incubate at room temp. for 5 min.

3. Remove fixative. (OPTIONAL: rinse with PBS, 2 x 1 ml).

4..Add 0.5 ml X-gal stain, place cells at 37°C for 30 min to several hours.

Materials and Reagents

Fixative: 0.05 % glutaraldehyde in PBS (for 10 ml, add 20 ul 25 % glutaraldehyde from Sigma to 10 ml PBS).

X-gal stain (prepare just before use):

Volume for 5 ml Final Concentration

500 ul 1 M NaPO₄ pH 7.3 100 mM

300 ul 50 mM K₃Fe(CN)6 3 mM

300 ul 50 mM K₄Fe(CN)₆ 3 mM

6.5 ul 1 M MgCl₂ 1.3mM

3.6 ml H2O

250 ul X-gal 1 mg/ml

- X-gal is kept at 20mg/ml stock in dimethylformamide at -20°

- Potassium ferricyanide stocks are stored at 4°