Order 3, Get Your 4th Free!* | Learn More >>
9860
Senescence β-Galactosidase Staining Kit
Cellular Assay Kits
Assay Kit

Senescence β-Galactosidase Staining Kit #9860

Reviews ()
Citations (63)
Senescence β-Galactosidase Staining Kit: Image 1

β-Galactosidase staining at pH 6 on normal WI38 cells at population doubling 29 (left) and senescent WI38 cells at population doubling 36 (right).

Senescence β-Galactosidase Staining Kit: Image 2

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).

To Purchase # 9860S
Product # Size Price
9860S
1 Kit  (125 35mm wells) $ 238

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

Protocol

PRINT

View >Collapse >

9860 Senescence β-Galactosidase Cell Staining Protocol

A. Solutions and Reagents

Reagents provided are sufficient to stain 125 x 35 mm wells.

Supplied Reagents

  1. 10X Fixative Solution #11674
  2. 10X Staining Solution #11675
  3. 100X Solution A #11676
  4. 100X Solution B #11677
  5. X-Gal #11678

Additional Reagents (Not Supplied)

  1. 1X PBS
  2. DMF (Dimethylformamide) #12767
  3. Polypropylene tubes
  4. 37°C dry incubator (No CO2)
  5. Phase contrast or light microscope
  6. 70% glycerol (optional)
  7. DMSO (Dimethyl Sulfoxide) #12611

B. Solution Preparation

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).

  1. PBS: Prepare at least 6 ml 1X PBS per 35 mm well
  2. Fixative Solution: Dilute the 10X Fixative Solution to a 1X solution with distilled water. You will need 1 ml of the 1X solution per 35 mm well.
  3. Staining Solution: Redissolve the 10X Staining Solution by heating to 37°C with agitation. Dilute the 10X staining solution to a 1X solution with distilled water. You will need 930 ul of the 1X Staining Solution per 35 mm well.
  4. X-Gal:

    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.

  5. β-Galactosidase Staining Solution: For each 35 mm well to be stained, combine the following in a polypropylene container:
    1. 930 µl 1X Staining Solution (See step 3)
    2. 10 µl 100X Solution A
    3. 10 µl 100X Solution B
    4. 50 µl 20mg/ml X-gal stock solution (see Step 4)

    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.

C. Procedure

  1. Remove growth media from the cells.
  2. Rinse the plate one time with 1X PBS (2 ml or a 35 mm well plate, or match volume of media)
  3. Add 1 ml of 1X Fixative Solution to each 35 mm well. Allow cells to fix for 10-15 min at room temperature.
  4. Rinse the plate two times with 1X PBS (SAFE STOP) Alternatively, plates can be left in 1X PBS, covered at +4°C overnight.
  5. Add 1 ml of the β-Galactosidase Staining Solution to each 35 mm well (see Solution Preparation Step 5).
  6. Important: Seal plate with parafilm to prevent evaporation. Evaporation can cause crystals to form.

  7. Incubate the plate at 37°C at least overnight in a dry incubator (no CO2).
  8. Note: The presence of CO2 can cause changes to the pH which may affect staining results.

  9. While the β-galactosidase is still on the plate, check the cells under a microscope (200X total magnification) for the development of blue color.
  10. For long-term storage of the plates, remove the β-Galactosidase staining solution and overlay the cells with 70% glycerol. Store at 4°C.

posted August 2020

Protocol Id: 2164

Product Description

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.

Background

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.

  1. Goldstein, S. (1990) Science 249, 1129-33.
  2. Sherwood, S. W. et al. (1988) Proc. Natl. Acad. Sci., USA 85, 9086-90.
  3. Dimri, G. et al. (1995) Proc. Natl. Acad. Sci., USA 92, 9363-7.
  4. Cristofalo, V. J. et al. (1998) Crit. Rev. Eukaryot Gene Expr. 8, 43-80.
  5. Linskens, M. H. et al. (1995) Nucleic Acid Res. 23, 3244-51.

Pathways & Proteins

Explore pathways + proteins related to this product.

Product Citations: 63

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.