Beta-gal staining of eukaryotic cells in vitro

 

(Modification of methods of Dr. Seong-Seng Tan and Promega's "Protocols and Applications Guide")

Cells previously transfected with a lac Z construct can be stained for b-galactosidase activity.

1. Wash cells 2 - 3 x with PBS (2 ml for 35 mm dish; I use room temp. tissue culture PBS without Ca++ or Mg++) - beware that some cell types, eg. NIH-3T3, BALB/C-3T3 or morphologically transformed cells are easily sloughed off the surface after 2nd wash.

2. Aspirate.

3. Fix cells with 0.2% glutaraldehyde/PBS for 5 min at RT (1 ml/35 mm dish). Note that glutaraldehyde vapor is quite toxic and should be diluted in fume hood. We use EM grade glutaraldehyde (Merck #4239, 25%) but lesser grade may be OK. Overfixing (longer times or higher % will reduce the signal).

4. Aspirate after 5' and then wash 2 x 2 ml PBS. Aspirate and add b-gal. stain.

Recipe for X-gal soln (made up in PBS)

Stock Solution Final Concentration To make 2 ml

X-gal (20 mg/ml in dimethylformamide) 1 mg/ml 100ul

potassium ferricyanide (0.5 M made in sterile dH20) 5 mM 20ul

potassium ferrocyanide (0.5 M made in sterile dH20 5 mM 20ul

MgCl2 (1 M) 2 mM 4ul

PBS 1.856 ml

Prepare just b/f use.

7. Incubate 37�, humidified incubator (T/C) for 2h - o/n. NOTE: The plate will dry out quickly if incubated in normal oven.

8. All +ve cells should stain blue (shades of from deep, royal, to light) within 2h, background shows up a/f 3-4 days incubation in X-gal soln (should include a no DNA transfected control).

Summary of 7 quick steps to get the Blues.

1. Wash cells 2-3 x 2 ml PBS

2. Aspirate off supernatant

3. Fix cells at RT with 0.2% glutaraldehyde 5 min (1:125 of a 25% solution).

4. Aspirate off glutaraldehyde

5. Wash 2 x 2 ml PBS. Aspirate

6. Stain for b-gal with X-gal soln.

7. Incubate 37�, 5% CO2 2h - o/n