Cell Meter™ Cellular Senescence Activity Assay Kit *Green Fluorescence*

1. Treat samples as desired
2. Prepare and add Xite™ beta-D-galactopyranoside working solution to samples
3. Incubate samples at 37 °C for 15 to 45 minutes
4. Monitor the fluorescence intensity using flow cytometer with 530/30 nm filter (FITC channel)

Thaw each kit component at room temperature before starting the experiment.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 100 uL DMSO (Component C) into Xite™ beta-D-galactopyranoside (Component A) and mix well. Note: Store the unused Xite™ beta-D-galactopyranoside stock solution at -20 °C in single use aliquots.

Dilute 10 uL of Xite™ beta-D-galactopyranoside stock solution (100X) with 1 mL of Assay Buffer to make Xite™ beta-D-galactopyranoside working solution (1X). Note: Xite™ beta-D-galactopyranoside working solution should be used promptly.

1. Treat your samples as desired.

   Note: For 96 well plate format, grow cells in 100 µL of cell culture medium and treat as desired. Volumes can be adjusted based on the plate size.

2. Wash the cells with buffer of your choice such as DPBS. 

   Note: For selectively tracking β-Gal in live cells, cells can be treated with Bafilomycin A1 for blocking endogenous β-Gal. Optimum concentration of Bafilomycin A1 may vary on type of cells.

3. Add 100 µL Xite™ beta-D-galactopyranoside working solution for 15-45 minutes and incubate the samples at 37°C incubator.

   Note: Optimal time for incubation needs to be determined carefully.

4. Remove the working solution and wash cells with buffer of your choice.

   Note: If performing flow cytometry for attached cells, cells can be trypsined at this step and collected in a tube.

5. Resuspend the cells in the Assay Buffer (Component B) and monitor the fluorescence intensity with flow cytometer using 530/30 nm filter (FITC channel) or fluorescence microscope with FITC filter set.