UA-Glo® 3D Cell Viability Assay

The 3D Cell Viability Assay Kit is designed for quantitatively measuring ATP levels in 3D model cells, where ATP content is directly proportional to the number of viable cells. This reagent features high signal-to-noise ratio, excellent reproducibility, and superior stability. Its ready-to-use formulation eliminates the need for separate lysis and detection steps, thereby minimizing errors caused by frequent pipetting. Additionally, its stable luminescent signal makes it particularly suitable for high-throughput 3D model cell proliferation assays and compound screening.

After mixing luciferase, luciferin, and buffer, the solution is filled into 10 ml or 100 ml brown bottles with the following specifications:

1. Cell Preparation
1) 3D Cell Model Construction: Construct relevant 3D cell models according to experimental requirements, such as 3D spheroid models.
2) If the experiment involves testing the effects of compounds on 3D cell models, add the test compounds at appropriate concentrations to the cell plate wells. Maintain the organic solvent concentration in the culture medium below 2%. Continue culturing for an appropriate duration based on experimental needs.

2. Cell Viability Assay
1) Take out the 3D cell viability assay reagent [Note 1] and equilibrate it to room temperature (22°C-25°C) [Note 2]. Gently shake to mix.
2) Remove the cell culture plate to be tested and equilibrate it to room temperature (22°C-25°C). The 3D cell viability assay should be performed in a white assay plate. If the cell culture plate is transparent or black, transfer the 3D model to a white assay plate before testing.
3) Add 100 µL of the 3D cell viability assay reagent to 100 µL of the 3D model cells in a 96-well plate, or 20 µL of the reagent to 20 µL of the 3D model cells in a 384-well plate [Note 3].
4) Vigorously shake the plate for 5 minutes to fully lyse the cells [Note 4], then place it in the dark to equilibrate the reaction for 25 minutes.
5) Measure the fluorescence signal using a fluorescence plate reader [Note 5].

1) After first use, the reagent should be aliquoted and stored protected from light at -20°C or below to ensure stability. The 3D cell viability assay reagent shows less than 10% signal reduction after 5 freeze-thaw cycles with no functional loss. When stored at room temperature (22°C) for 8 hours or at 4°C for 72 hours, the signal reduction is less than 10% with no functional loss.2) The luciferase reaction in the 3D cell viability assay reagent is sensitive to temperature changes. Both the reagent and assay plate must be equilibrated to room temperature (22°C-25°C), and the temperature should remain constant (±1°C) during testing.3) Unless rigorously validated, arbitrary changes to the reagent volume are not recommended. The volume ratio of cell culture medium to assay reagent should be 1:1.4) Vigorous shaking of the assay plate to ensure complete cell lysis is essential, while avoiding spillage of liquid from the wells.5) The signal decay rate of the 3D cell viability assay reagent varies across different 3D cell models, with a half-life ranging from 1.5h to 3h. It is recommended to complete plate reading within 1h-2h.6) For 3D cells cultured for more than 7 days: After adding the reagent, ensure extended incubation time with continuous plate shaking at 37°C to achieve thorough lysis.7) This product is for research use only.