LacZ staining of cells

The bacterial enzyme &beta;-galactosidase (gene LacZ) is frequently used as reporter gene. It can be easily located with a LacZ stain using the artificial substrate X-gal, which turns blue when it is cleaved by &beta;-galactosidase.

This protocol describes the staining of cultured cells containing active LacZ genes.

material

 * X-gal stock is 200 mg/ml in dimethylformamide (DMF)
 * K ferri-cyanide ( 50mM ) =Fe3- is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
 * K ferro-cyanide ( 50mM ) =Fe4- is 0,42 g / 20 ml PBS ( Sigma Cat.# P-9387)

staining solution

 * make fresh
 * heat mix to 50°C before adding X-Gal, in order to avoid precipitation

fixation solution

 * make fresh

common mistakes / tips

 * time of fixation is crucial; overfixing cells will reduce LacZ activity
 * some cell types are easy to detach by squirting liquid onto the dish; be careful;
 * X-Gal will precipitate if solution cools too much

steps
(volume is around 5 ml for 10 cm-dish                 3 ml for 6 cm-dish			                 500µl for a 24 well)
 * 1) carefully wash cells once with PBS (avoid detaching cells by strong pipetting)
 * 1) add fixative solution
 * 2) incubate 2 min (Time is important !)
 * 3) carefully wash 3 times with PBS (1st wash must be quick to avoid overfixation in the last samples to be washed)
 * 4) add staining solution
 * 5) incubate over night at 37°C