Senescence β-Galactosidase Staining Kit #9860

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).

Important: Seal plate with parafilm to prevent evaporation. Evaporation can cause crystals to form.

6. Incubate the plate at 37°C at least overnight in a dry incubator (no CO₂).

Note: The presence of CO₂ can cause changes to the pH which may affect staining results.

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