Description

Cell Senescence Assay

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 ScienCellT 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

Cat. No	# of Vials	Reagents	Quantity	Storage
8058a	1	Staining solution A	1 ml	40C
8058b	1	Staining solution B	1 ml	40C
8058c	1	Staining solution C	0.2 ml	40C
8058d	1	Staining solution D	20 ml	40C
8058e	1	X-Gal solution	5 ml	-200C
8058f	1	100X Fixing solution	1 ml	40C

Quality Control

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

Procedures

1. Preparation of Reagents
1. Preparation of working fixing solution: Prepare 1× 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
   +  1856 µl of Staining Solution D
   -------------------------------------------
     2000 µl of working staining solution
2. 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.

* Crystal deposition, which comes from unreacted X-gal, may be observed after incubation of cells with working staining solution. It can be minimized by pre-filtering the working staining solution with a 0.2 µm filter.