-Galactosidase Staining of Eukaryotic Cells in vitro

Saturday, October 18, 2003

Description
Cells that were previously transfected with a lac Z construct can be stained for -Galactosidase activity.

Procedure
1. Wash the cells 2 to 3 times in 2 ml of PBS solution (see Hint #2 and Hint #3).

2. Aspirate the supernatant.

3. To the cells add 1 ml of 0.2% Glutaraldehyde per 35 mm dish.

4. Incubate at room temperature for 5 min.

5. Aspirate the Glutaraldehyde and wash the cells 2 times in 2 ml PBS solution (see Hint #3).

6. Aspirate the PBS solution and add the -Gal Stain Solution (1 ml per 35 mm dish, see Hint #4).

7. Incubate at 37C in humidified incubator (5% CO2) from 2 hours to overnight (see Hint #5).

8. All positive cells should stain blue (shades from deep blue, royal blue to light blue) within 2 hours. Background staining is seen after 3 to 4 days of incubation in -Gal Stain Solution.

9. Aspirate the supernatant and wash in PBS.

Recipes
500 mM Potassium Ferrocyanide (CAUTION, see Hint #1)

-Gal Stain Solution

500 mM Potassium Ferricyanide (CAUTION, see Hint #1)

-Gal Stain Solution

X-Gal Solution (CAUTION, see Hint #1)
Prepared in Dimethylformamide
20 mg/ml X-Gal

Glutaraldehyde Solution 0.2% (v/v) Glutaraldehyde (Electron Microscopy Grade)
Prepared in PBS solution
(CAUTION, see Hint #1)

PBS pH 7.2
137 mM NACl
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)

-Gal Stain Solution

-Gal Stain Solution

-Gal Stain Solution 20 l Potassium Ferricyanide
20 l Potassium Ferrocyanide
To prepare 2 ml:
Mix well and prepare in a fume hood just before use.
100 l X-Gal solution
1.856 ml PBS

-Gal Stain Solution

1 M MgCl2

Supplies

Tips
1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Make sure to include a control cell group that was not transfected with a lac Z construct.

3. Beware that some cell types (NIH-3T3, BALB/C-3T3 or morphologically transformed cells) will easily slough off the surface after the second wash. During aspiration you could lose these cells if you do not include a step to pellet the cells before aspiration.

4. Over fixing (such as longer times or increased percent glutaraldehyde) will reduce the signal.

5. Plates will dry out if incubated in a standard oven.

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