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

1. Treat samples as desired

2. Prepare and add Xite™ Red beta-D-galactopyranoside working solution to samples

3. Incubate samples at 37 °C for 15 to 45 minutes

4. Monitor the fluorescence intensity with fluorescence microscope using Cy3/TRITC filter set or flow cytometer using 575/26 nm filter (PE channel)

To ensure accurate results, allow all kit components to reach room temperature before beginning 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 µL of DMSO (Component C) into Xite™ Red beta-D-galactopyranoside (Component A) and mix thoroughly.

Note: Divide any remaining Xite™ Red beta-D-galactopyranoside stock solution into individual single-use aliquots. Store these aliquots at -20°C, protected from light.

To prepare the Xite™ Red beta-D-galactopyranoside working solution, mix 10 µL of the Xite™ Red beta-D-galactopyranoside stock solution with 1 mL of Assay Buffer.

Note: Xite™ Red 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. Remove the cell culture medium and wash the cells with a 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. The optimum concentration of Bafilomycin A1 may vary on the type of cells.

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

   Note: The 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 a flow cytometer using a 575/26 nm filter (PE channel) or fluorescence microscope using a Cy3/TRITC filter set.