Senescence β-Galactosidase Activity Assay Kit (Fluorescence, Plate-Based) #23833

Senescence β-Galactosidase Activity Assay Kit - Plate Based Protocol

A. Solutions and Reagents

Supplied Reagents

1. 1X Senescence Cell Lysis Buffer: (#91029)
2. 2X Senescence Reaction Buffer: (#78494)
3. SA-β-Gal Substrate: (#45954) Reconstitute vial of SA-β-Gal Substrate in 348 µL of DMSO to make a 20X solution. Store any unused substrate at -20°C for up to 1 month from date of reconstitution.
4. Senescence Stop Solution: (#66008)

Additional Reagents (Not Supplied)

1. β-mercaptoethanol (BME)
2. DMSO (Dimethyl Sulfoxide) Sterile: (#12611)
3. PMSF: (#8553) Reconstitute 34.84 mg of lyophilized PMSF in 1.0 ml isopropyl alcohol, to make a 200 mM solution.
4. Protease/Phosphatase Inhibitor Cocktail (100X): (#5872)
5. Phosphate Buffered Saline (PBS-20X): (#9808) To prepare a 1X solution, add 0.5 mL of 20X PBS to 9.5 mL of deionized water.
6. 96-well plate for initial reaction found in Section C. Test Procedure Step 1.
7. 96-well black opaque plate suitable for a fluorescent plate reader.
8. Plate reader capable of reading excitation at 360 nm and emission at 465 nm.
9. BCA Protein Assay Kit: (#7780)
10. High Speed Centrifuge

Reagent	96-well	24-well	6-well	10cm dish
1X PBS Wash	100 µL	400 µL	1000 µL	1500 µL
1X Cell Lysis Buffer	100 µL	400 µL	1000 µL	1500 µL

B. Preparing Cell Lysates

NOTE: Cell treatments to induce senescence should be completed before initiating assay with this kit. Ensure that an untreated control is included, to provide a baseline measurement of fluorescence for comparison.

1. Plate cells of interest in a tissue culture vessel and culture under appropriate conditions used to generate senescent cells.
2. Prepare a 1X Senescence Cell Lysis Buffer immediately before use; add the proper amount of protease inhibitor such as 1.0 mM PMSF and protease/phosphatase inhibitor cocktail to the solution. Keep solution on ice while making the cell lysate. Refer to the provided table to calculate how much volume is needed based on the size of the cell culture vessel.
3. Aspirate the medium from the vessel containing cells being analyzed.
4. Wash the cells once with cold 1X PBS (refer to the table for correct volumes), then aspirate and discard the wash.
5. Add cold 1X Senescence Cell Lysis Buffer (refer to Section B. Step 2) to the cells (refer to the table for correct volumes). Incubate on ice for 5 minutes.
6. Scrape the cells off the surface and transfer the whole lysate to a micro-centrifuge tube. Pipette the lysate up and down several times to facilitate the lysing process.
7. Place the micro-centrifuge tubes containing the cell lysate into a centrifuge and spin for 5 minutes (x14000 rpm) at 4°C.
8. Transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.
9. Optional, determine the total protein concentration of each cell lysate sample by protein assay.

C. Test Procedure

2X Assay Buffer Preparation: Immediately before use, add BME to 2X Senescence Reaction Buffer at a final concentration of 10 mM. Then dilute the 20X SA-β-Gal Substrate to 1X with 2X Senescence Reaction Buffer containing 10mM BME. Make only enough solution for the number of wells analyzed (50 µL per well). Use the 2X Assay Buffer immediately and properly discard any unused solution.

1. Transfer 50 µL of the cell lysate to a 96-well plate. Add 50 µL of freshly prepared 2X Assay Buffer. Incubate the samples at 37°C protected from light for 1-3 hours.
2. Remove 50 µL of the reaction mixture and transfer to a 96 well black opaque plate.
3. Stop the reaction by adding 200 µL of Senescence Stop Solution to each well.
4. Read fluorescence with a fluorescence plate reader set with excitation at 360nm and emission at 465nm. For optimal readings, read the plate within 30 minutes of adding STOP solution.