Beta-Galactosidase Senescence Assay

Principles:

Using X-gal blue developing to detect the cell senescence.(Search Senescence Assay in google for details.)

Reagents

1. Glutaraldehyde fixative (25%) (1 ml). Store at -20^oC.

Glutaraldehyde fixative (0.05%): It is supplied as a 25% solution. Store frozen at -20^oC. It can be frozen and thawed many times. Immediately before use, thaw the vial and dilute the required amount to 500-fold in PBS to 0.05%.

Caution: Glutarldehyde is a carcinogen and may cause allergic reaction. Use it in a fume hood and discard waste as required by your institution.

2. 3% formaldehyde

3. X-Gal Stock＝20 mg/ml in dimethyl-formamide, -20℃ store in darkness.

4. 0.1M K3Fe(CN)6 (potassium ferricyanide) (5 ml). Store at room temperature for 6 months.

5. 0.1M K4Fe(CN)6.3H2O (potassium ferrocyanide) (5 ml). Store at room temperature for 6 months.

6. 1M MgCl2. (1 ml). Store at room temperature.

7. X-Gal solution:

X-gal 1 mg/ml

Citric acid/sodium phosphate 40 mM, pH 6.0

Potassium ferrocyanide（K₄Fe[CN]₆, )5 mM

Potassium ferricyanide（K₃Fe[CN]₆,） 5 mM

NaCl 150 mM

MgCl₂ 2 mM

OR: Prepare in PBS from the stock solutions as follows:

5 to 35 mM 0.1M K3Fe(CN)6 (potassium ferricyanide)

5 to 35 mM 0.1M K4Fe(CN)6.3H2O (potassium ferrocyanide)

1 to 2 mM MgCl2

Just before use add X-gal (20 mg/ml) in DMF to 1 mg/ml final concentration.

Note: The amount of ferric or ferrous cyanide to use can be determined by trial and error. The higher amount causes precipitation of indole to occur more rapidly thus reduces diffusion.

Procedure

1． Cell were washed in PBS (pH 7.2)；

2． Fixed for 3-5 min at RT in 2% formaldehyde/0.2% glutaraldehyde(or 3% formaldehyde)，ORFix with 0.5% glutaradehyde (in PBS pH 7.2) for 5 min，OR Add fixative solution (0.05% glutarldehyde in PBS) (2 ml for 6-cm dish and 5 ml for 10-cm dish). Alternatively, cells can be grown on glass chamber slides (Lab-Tek). Incubate at room temperature for 5 to 15 min.

3． Discard fixative solution using proper chemical disposal protocol for your institution, and rinse cells 1 x in PBS followed by 10 minutes wash in PBS and another quick rinse in PBS. These washings are done at room temperature.

4． Add X-gal solution just to cover the cells. Incubate 1 hr to overnight at 37oC (no CO₂).

5． Staining was evident in 2-4 hr and maximal in 12-16 hr. Positive stains will stain blue. Blue color seen by naked eye is an indication of strong staining.

6． Photograph by microscope at 100×or 200×.

NOTES：

1. Longer fixation may inhibit enzyme activity in subsequent steps.

2. It is important to wash the fixative thoroughly; otherwise it may inhibit the enzyme reaction.

3. X-gal solution should be fresh.

4. The stained cells can be stored indefinitely at 4^oC.

5. Proper sterile techniques should be used. All incubations involving tissue culture cells should be done in a humidified 37%, 5-10% CO2 incubator.

6. It is best to perform all steps involving fixatives in a fume hood.

7. Avoid contact and inhalation of cyanide. Discard waste as required.

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