Cellular senescence is a state where cells can no longer divide, despite the presence of appropriate growth factors. This state can be induced either by telomere shortening (replicative senescence) or by telomere-independent signals, such as deoxyribonucleic acid (DNA) damage and oxidative stress (stress-induced senescence). The state of cellular senescence is associated with the following: irreversible growth arrest; a distinct flat, and enlarged cell morphology; an increase in senescence-associated ß-galactosidase (SA ß-gal) activity; and the expression of cell cycle inhibitors, such as p16^INK4a and p21^{CIP1/WAF1/Sdi1}

BioPioneer providesasimple andrapid test for identifying senescent cells and gives science a powerful new tool for examining senescence. This kit is designed to detect SA-beta-galactosidase which catalyzes the hydrolysis of X-gal and produces a blue color in senescence at pH6.0.

Kit Contents:

10X Fixative Solution 15 ml

X-Gal 150 mg

10X Staining Solution 15 ml

100X Staining Solution Supplement A: 1.5ml

100X Staining Solution Supplement B:1.5ml

Materials Supplied by the User

• 1X PBS (Phosphate Buffered Saline):
• N-N-dimethylformamide (DMF)
• 50% glycerol (optional)
• 37°C incubator
• Phase contrast or light microscope
• Polypropylene tubes

Solution Setup

The following protocol is designed for one 35 mm well of a 6-well plate. (1 ml for 35 mm wells/plates, 2.5 ml for 60 mm plates and 5 ml for 100 mm plates).

Polypropulene tubes must be used duringsolution preparation.

1. Prepare a 6ml 1X PBS solution for each 35mm well (not provided).

2. Dilute the 10X Staining and 10X Fixative Solutions with distilled water to make 1X solutions. You will need 930 ul of 1X Staining Solution and 1 ml of 1X Fixative Solution per 35 mm well.

3. Dissolve 20 mg X-gal in 1 ml DMF to prepare a 20X stock solution. Excess X-gal solution can be stored at -20°C in a light resistant container for one month.

Assay Protocol

1. Aspiratethe growth medium from the cells and wash the plate once with 2 ml 1X PBS.

2. Add 1 ml 1X Fixative Solution to the cellsand incubate for 5-10 minutes at room temperature.

3. During the fixingprocess , preparethe Staining Solution.

a. 930 ul Staining Solution

b. 10 ul Staining Supplement A

c. 10 ul Staining Supplement B

d. 50 ul 20 mg/ml X-gal in DMF

4. Remove the fixing solution and wash the fixed cells twice with 2 mL of 1X PBS.

5. Add 1 ml Staining Solution mix to the plate. Incubate 5 hours toovernight at 37°C.

6. Check the cells under a microscope (200 x total magnification) for development of blue color.