β-Galactosidase staining at pH 6 on normal WI38 cells at population doubling 29 (left) and senescent WI38 cells at population doubling 36 (right).
Β-Galactosidase staining at pH 6.0 on MCF-7 cells untreated (left) and senescent MCF-7 cells treated with etoposide #2200 (12.5 μM, 24 hr) and allowed to recover for 4 days (right).
|Product Includes||Quantity (with Count)|
|10X Fixative Solution||1 x 15 ml|
|10X Staining Solution||1 x 15 ml|
|100X Solution A||1 x 1.5 ml|
|100X Solution B||1 x 1.5 ml|
|X-Gal||1 x 150 mg|
Reagents provided are sufficient to stain 125 x 35 mm wells.
Additional Reagents (Not Supplied)
NOTE: All solutions should be prepared just prior to use.
Volumes are for one 35 mm well of a 6-well plate. Volumes in the procedure should be approximately half that of the tissue culture media. (e.g. 1 ml for 35 mm well/plates containing 2 ml of media, 2.5 ml for 60 mm plates containing 5 ml of media, and 5 ml for 100 mm plates containing 10 ml of media).
IMPORTANT: Always use polypropylene plastic or glass to make and store X-gal. Do not use polystyrene.Dissolve 20 mg of X-gal in 1 ml DMF to prepare a 20 mg/ml stock solution. Excess X-gal solution can be stored in -20°C in a light resistant container for up to one month.
Note: DMSO can be used in place of DMF.
IMPORTANT: Due to variations in water pH, please be sure that the β-Galactosidase Staining Solution has a final pH of 6.0 (A pH 5.9-6.1 is acceptable). pH differences can affect staining: A low pH can result in false positives and high pH can result in false negatives. If necessary, use HCl or NaOH to lower or raise pH, respectively.
Important: Seal plate with parafilm to prevent evaporation. Evaporation can cause crystals to form.
Note: The presence of CO2 can cause changes to the pH which may affect staining results.
posted August 2020
Protocol Id: 2164
The Senescence β-Galactosidase Staining Kit is designed to conveniently provide reagents needed to detect β-galactosidase activity at pH 6, a known characteristic of senescent cells. Papers have published using this kit in both cells and frozen tissue. The kit includes all reagents necessary for this assay.
Limited capacity to replicate is a defining characteristic of most normal cells and culminates in senescence, an arrested state in which the cell remains viable (1). Senescent cells are not stimulated to divide by serum or passage in culture, and senescence invokes a specific cell cycle profile that differs from most damage-induced arrest processes or contact inhibition (2). An enlarged cell size, expression of pH-dependent β-galactosidase activity (3), and an altered pattern of gene expression (4,5) further characterize senescent cells.
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