Introduction
Normal mammalian cells divide for a limited number of population doublings and eventually enter an arrested state in which the cells remain alive, but do not proliferate in response to mitogens, and assume a characteristic enlarged, flattened morphology. This process, termed senescence, is thought to be a tumor suppressive mechanism and underlying cause of aging. Senescence-associated beta-galactosidase (SA- beta-gal) is a widely used biochemical maker for assessing senescence in cultured cells. The ScienCell™ Cell Senescence Assay provides an easy-to-use method to detect SA- beta-gal by staining cells with 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) at pH 6.0, a pH condition that suppress lysosomal beta-galactosidase activity sufficiently to ensure that nonsenescent cells remain unstained.

Kit Components

Quality Control
The Cell Senescence Assays are applied to early passage and senescent ScienCell™ Human Renal Proximal Tubular Epithelial Cells (HRPTEpiCs). Data show that the senescent cells show positive SA-beta-gal staining while most early passage cells don't (Figure 1).

Procedure
A. Preparation of reagents
1. Preparation of working fixing solution: Prepare 1X fixing solution by diluting 100X Fixing Solution stock 1:100 in PBS.
2. Preparation of working staining solution: Prepare fresh staining working solution based on the number of samples to be assessed. For each sample in 35 mm plate, prepare the following mixture:

100 µl of X-gal Solution
20 µl of Staining Solution A
20 µl of Staining Solution B
4 µl of Staining Solution C
400 µl of Staining Solution D
1456 µl of DI H2O

Total of 2000 µl of working staining solution

B. Staining protocol
1. Remove culture medium from cells and rinse twice with PBS.
2. Fix cells by incubating with 2 ml of working fixing solution for 3-5 minutes at room temperature.
3. Aspirate working fixing solution and rinse the fixed cells three times with PBS.
4. Add 2 ml of working staining solution to completely cover cells and incubate cells at 37°C, protected from light, for 12-24 hours, blue color should develop in senescent cells. Examine cells at regular time points to avoid overstraining.
5. After incubation, remove working staining solution and rinse cells twice with PBS, keep the cells in PBS at 4°C. Examine and count the blue stained cells using a light microscope.

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