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
