Senescence Cells Histochemical Staining Kit
SIGMA Catalog # CS0030
1. Before use, thaw all kit components and mix thoroughly until the
solutions are homogenous.
2. Ultrapure water (17 MΩ‧cm or equivalent) water should be used to
prepare the working solutions for this kit.
3. X-gal Solution - Warm the X-gal Solution at 37 ?C for 1 hour. Warming
this solution is very important to avoid formation of aggregates that
could interfere with the visualization of the stained cells. 4. 1× Fixation Buffer - Dilute the Fixation Buffer 10-fold with ultrapure
water. Use of 0.2 mm filtered water is recommended. After
preparation the 1× Fixation Buffer should be stored at –20 ?C. Prepare
10 ml for 6 tests in a 35 mm plate or one 6 well plate. 5. 1× PBS - Dilute the PBS 10-fold with 0.2 mm filtered ultrapure water.
After preparation, the 1× PBS should be stored at 2–8 ?C. Prepare 35 ml
for 6 tests in a 35 mm plate or one 6 well plate. It is possible to prepare
larger volumes and store the 1× PBS at 2–8 ?C.
6. Staining Mixture - (Prepare just prior to use),Mix the following for
preparation of 10 ml of the Staining Mixture:
1 ml of Staining Solution 10×
125 ul of Reagent B
125 ul of Reagent C
0.25 ml of X-gal Solution
8.50 ml of ultrapure water
Filter the Staining Mixture using a 0.2 mm filter to ensure there are no
aggregates in the solution.
1. Aspirate the growth medium from the cells.
2. Wash the cells twice with 1 ml of 1× PBS per well/plate. Carefully
remove the entire wash solution by aspiration, so the cells do not
3. Add 1.5 ml per well of 1× Fixation Buffer and incubate the plate for 6–7
minutes at room temperature.
4. During the fixation process prepare the Staining Mixture as described in
the Preparation Instructions.
5. Rinse the cells 3 times with 1 ml of 1×PBS per well/plate.