Amplite® Fluorimetric Beta-Galactosidase Assay Kit *Red Fluorescence*

Protocol Summary

1. Prepare stable or transient transfected cells with LacZ gene
2. Incubate cells (samples) with test compounds
3. Lyse the cells
4. Transfer the lysate to a microtiter plate
5. Add β-Gal working solution
6. Incubate at room temperature or 37°C for at least 10 minutes depending on cell type
7. Add stopping solution
8. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components to room temperature before use. 

1. β-Galactosidase Substrate stock solution (200X):
Add 50 µL of DMSO (Component E) into the vial of Resorufin β-Galactosidase (Component A) to make 200X β-Galactosidase Substrate stock solution. Note: 25 µL of β-Galactosidase Substrate stock solution is enough for 1 plate. Keep from light.

1. 0.3 % β-mercaptoethanol assay buffer:
Add 30 µL of β-mercaptoethanol (Component F) to 10 mL of Reaction Buffer (Component B), and mix well. Note: Additional buffer is needed for preparing enzyme dilution buffer, which is used to generate a standard curve.

2. β-Gal working solution:
Add 25 µL of β-Galactosidase Substrate stock solution (200X) into 5 mL of 0.3 % β-mercaptoethanol assay buffer. Note: β-Gal working solution is enough for one 96-well plate.

3. Lysis buffer working solution:
Add 5 µL of β-mercaptoethanol (Component F) to 5 mL of Lysis Buffer (Component D) before use. Note: Always add 0.1% β-mercaptoethanol into lysis buffer before lysing the cells

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Table 1. Recommended Lysis Buffer working solution volumes for cell culture plates.

Prepare cell extracts from mammalian cells

1. Treat cells containing LacZ gene with test compounds for a desired period of time.
   
   
2. Wash the cells twice with 1X PBS. Do not dislodge the cells.
   
   
3. Lyse cells accordingly with Lysis Buffer working solution.
   
   For adherent cells: Add Lysis Buffer working solution to the culture plates. See table 1 for recommended volumes.
   
   For non-adherent cells: Pellet the cells into centrifuge tube, and add 50 - 2000 µL (depending on the size of the cell pellet) of Lysis Buffer working solution to the tube.
   
   
4. Incubate cells from previous step at room temperature for 10 - 15 minutes, and gently swirl the plates or tubes several times to ensure complete lysis.
   
   
5. Proceed to the β-Galactosidase assay or freeze the sample at -80 °C until use. Note: A good lysis can also be obtained by a quick freeze-and-thaw cycle (freeze 1 - 2 hours at -20°C to -80°C and thaw at room temperature). Alternatively, centrifuge the cell lysis for 2 - 3 minutes to pellet the insoluble material, and then assay the supernatant.
   
   

Run ß-galactosidase assay

1. Thaw the tube or plate of lysed cells at room temperature if needed. Perform the assay directly on the 96-well plate if the cells were seeded in a 96-well plate.
   
   
2. Add 50 µL of cell extracts into each well of the 96-well plate. Save some control wells for the standard curve (50 uL/well) if a standard curve is desired. Note: If necessary, dilute the lysate in Lysis Buffer working solution when transfection efficiency is very high or reduce the volume of lysis buffer when transfection efficiency is low. If the transfection is performed in a 96-well plate, or a stable cell line was seeded into a 96-well plate, perform the assay directly on the plate. For endogenous β-galactosidase activity control, add 50 µL of cell lysate from non-transfected cells. For blank control, add 50 µL of Lysis Buffer working solution.
   
   
3. Add 50 µL of ß-Gal working solution to each well. Incubate the plate at room temperature or 37 °C for approximately 10 min to 4 hr depending on the cell type.
   
   
4. Add 50 µL of Stop Buffer (Component C) to each well. The stop buffer causes an increase in the fluorescence intensity of the product, in addition to terminate the reaction.
   
   
5. Measure the fluorescence intensity of the solution in each well with a fluorescence microplate reader at Ex/Em = 540/590 nm (cutoff = 570 nm).