Endogenous β-galactosidase existing in living cells interfere with selective detection of SA-β-Gal. Baf. A1 is an inhibitor of ATPase in lysosome. pH in lysosome is kept neutral by adding Baf. A1. Cellular Senescence Detection Kit - SPiDER-βGal contains Baf. A1 which allows to detect SA-β-Gal selectively. Baf. A1 is utilized for living cell assays only. Baf. A1 is not used in fixed cells because intracellular pH is controlled with the buffer.
The figure above shows the difference in SA-β-Gal detection with and without Baf. A1. Baf. A1 is also known as an autophagy inhibitor. When using Baf. A1. on live cells, please consider whether there are any effects on the experiment and if so, we recommend experiments with fixed cells. Baf. A1 is not used with fixed cells as the intracellular pH is controlled by buffer. Fixed cells are stained with SPiDER-βGal as procedure described in the manual.

SPiDER-βGal DMSO stock solution and Bafilomycin A1 DMSO stock solution is stable for 1 month at -20 ℃.

SPiDER-βGal working solution and Bafilomycin A1 working solution can’t be stored. Be sure to use the working solution immediately.

Lipofuscin is a fluorescent pigment that accumulates in a variety of cell types with age. Lipofuscin consists of autofluorescent granules and may results in high background for fluorescence microscopy. In order to achieve accurate SA-β-gal activity assay in senescent cells, we recommend to prepare samples without SPiDER-βGal staining as a negative control. Please compare fluorescence intensity of both cells with or without SPiDER-βGal staining.

> For Flow Cytometry Detection

Step 1. Prepare senescent cells and non-senescent cells. Measure MFI (Mean Fluorescence Intensity) of samples below.

[Senescent cells]

Sample A: The cells stained with SPiDER-βGal

Sample B: The cells without SPiDER-βGal staining

[Non-senescent cells]

Sample A’: The cells stained with SPiDER-βGal

Sample B’: The cells without SPiDER-βGal staining

Step 2. Calculate SA-β-gal activity (senescent cells) with the following formula

SA-β-gal activity (senescent cells) = MFI of Sample A - MFI of Sample B

Step 3. Calculate SA-β-gal activity (non-senescent cells) with the following formula

SA-β-gal activity (non-senescent cells) = MFI of Sample A’ - MFI of Sample B’

• Determine the SA-β-gal activity by comparing the SA-β-gal activity between senescent cells and non-senescent cells.
• Change of SA-β-gal activity associated with senescence = (Value from Step 2- value from Step 3)

>For Microscopy

Step 1. Prepare senescent cells without SPiDER-βGal staining and observe fluorescent image.

Step 2. Adjust detection sensitivity in microscopy to reduce background autofluorescence of lipofuscin.

Step 3. Observe fluorescent image of senescent cells and non-senescent cells under the settled condition in step 2.

It is possible. If fixed cells are stained with SPiDER-βGal, it is not necessary to pre-treat the cells with bafilomycin A1. However, McIlvain buffer adjusted to pH 6.0 must be prepared separately. See manual for details.

See protocol below.

(1) Prepare cells in 35 mm dishes and incubate overnight at 37°C in a 5% CO2 incubator.
(2) After removing the culture medium, wash the cells once with 2 ml of HBSS. 
(3) Treat the cells with trypsin and mix with 500 µl serum containing medium to collect the cells.
(4) Centrifuge at 300 xg for 5 minutes and remove the supernatant.
(5) Suspend the cells in 100 µl 2% PFA/PBS to the cells and incubate for 5 minutes at room temperature.
(6) Centrifuge at 300 xg for 5 minutes and remove the supernatant.
(7) Suspend the cells in 500 µl HBSS, centrifuge at 300 ×g for 5 minutes and remove the supernatant. Repeat this step twice.
(8) Add 500 µL of SPiDER-βGal working solution (for fixed cells) and incubate for 30 minutes at 37°C.
　　 Note: Do not use a 5% CO2 incubator due to reduce pH fluctuations.
(9) Centrifuge at 300 xg for 5 minutes and remove the supernatant.
(10) Suspend the cells in 500 µl HBSS, centrifuge at 300 ×g for 5 minutes and remove the supernatant. Repeat this step twice.
(11) Suspend the cells in 500 µl HBSS and analyze on a flow cytometer.

Preparation of SPiDER-βGal working solution
Dilute the SPiDER-βGal DMSO stock solution 2,000 times ^*1 with McIlvaine buffer (pH 6.0).
*1 Fixation and permeablization could leads to lower sensitivity (Figure 1), if you need higher signals,dilute the SPiDER-βGal DMSO stock solution 500 – 1,000 times with the McIlvaine buffer (Figure 2).

Preparation of McIlvaine buffer (pH 6.0)
Mix 0.1 mol/l citric acid solution (3.7 ml) and 0.2 mol/l sodium phosphate solution (6.3 ml). Confirm the pH is 6.0. If the pH is not 6.0, adjust the pH by adding either citric acid solution or sodium phosphate solution. Dilute this buffer 5 times with ultrapure water.

Staining procedure (35 mm dish)
1. Prepare cells on 35 mm dish for assay and culture the dish at 37℃ overnight in a 5% CO₂ incubator.
2. Remove the culture medium. Add 2 ml of 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 3 minutes ^*2.
*2 Avoid a longer treatment period, which leads to decrease in SA-β-gal activity.
3. Remove the supernatant, and wash the cells 3 times with 2 ml of PBS.
4. Add 2 ml of SPiDER-βGal working solution and incubate at 37℃ for 30 minutes^*3.
*3 We recommend not to use a 5% CO₂ incubator for fixed cell experiments. If incubation is done in a 5% CO₂ incubator, the pH of the buffer may become acidic. Acidic pH results in higher background from the endogenous β-galactosidase activity and it would be difficult to distinguish between normal cells and senescent cells.
5. After removing the supernatant, wash the cells twice with PBS.
6. Add 0.1% Triton X-100/PBS to cells and incubate for 30 minutes at room temperature.
7. Wash the cells twice with PBS.
8. Add 1% BSA/PBS to the cells and incubate for 1 hour at the room temperature
9. Add anti- γ-H2AX antibody (mouse) diluted with 1% BSA/PBS to the cells and incubate at 4℃ overnight.
10. Wash the cells 3 times with PBS.
11. Add anti- mouse secondary antibody (Cy5) diluted with 1% BSA/PBS to the cells and incubate at room temperature for 1 hour.
12. Wash cells twice with PBS and observe under a fluorescence microscope.

Please check the following points.

(1) Use the filter that matches the fluorescence characteristics of the reagent.
　＜Recommended filters＞
　　Fluorescence microscope: excitation (500-540 nm), fluorescence (530-570 nm)
　　Flow cytometer: excitation (488 nm), fluorescence (500-540 nm)
　
(2) Use fresh working solution

(3) Increase the staining time.
　If fluorescence cannot be confirmed after 30 minutes of incubation after addition of SPiDER-βGal working solution, increase the incubation time to 45-60 minutes.

It is possible. Fixation with 4% paraformaldehyde is recommended.

Serum and phenol red in the medium do not affect the detection of SA-β-gal.

Cellular senescence wasn’t induced. Please prepare for positive control. Please visit product page and check “Positive Control”

The incubation with SPiDER-βGal working solution was done in a 5% CO2 incubator. We recommend not to use a 5% CO2 incubator during incubation with SPiDER-βGal working solution. If incubation is done in a 5% CO2 incubator, the pH of the buffer may become acidic. Acidic pH results in higher background from the endogenous β-galactosidase activity and it would be difficult to distinguish between control cells and senescent cells. Please incubate the plate in a dry incubator without CO2 .