Principle of Detection

 

The 2D Luminescent Cell Viability Assay relies on the ATP-dependent luminescence reaction catalyzed by luciferase to measure intracellular ATP levels through chemiluminescent signals, thereby detecting cell viability or quantifying the number of live cells. It features a wide linear range, high sensitivity, and good stability. In a 96-well plate, it exhibits a good linear relationship within the range of 100 to 100,000 cells, although the upper limit of detectable cell numbers may vary among different cell types. Moreover, the procedure is simple, with the detection reagents provided in the kit being ready-to-use, stable readings, fast detection speed, and the entire detection process taking only about 10 minutes. There is no need to wash the cells, nor is it necessary to replace or remove the culture medium. Compared with other common cell viability assays, such as Calcein-AM and CCK-8, the 2D Luminescent Cell Viability Assay is more simple and rapid.

 

 

Product Advantages

 

The 2D Luminescent Cell Viability Assay (abs50065) offers high sensitivity and a wide linear range, compatible with both small sample detection and high-throughput screening of large sample volumes.

1. Convenient and Rapid: The detection reagents provided in the kit are ready-to-use, with stable readings and fast detection speed, completing the assay in just about 10 minutes;

2. High Sensitivity: Capable of detecting as low as 10 nmol of ATP;

3. Wide Linear Range: In a 96-well plate, it demonstrates a good linear relationship within the range of 100 to 100,000 cells, though the upper limit of detectable cell numbers may vary among different cell types;

4. High-Throughput: Compatible with both small sample detection and high-throughput screening of large sample volumes.

 

Usage Method

 

I. Reagent Preparation

 

Thawing of Reagents: Remove the reagents from storage the day before the experiment and let them thaw overnight at 4℃. Alternatively, on the day of the experiment, take out the reagents and let them thaw at room temperature or in a 22℃ water bath, but ensure that the water temperature does not exceed 25℃.

Equilibration to Room Temperature: If the reagents have been thawed under non-room temperature conditions, place them in a 22℃ water bath before use to ensure they are equilibrated to room temperature before detection.

Note: Generally, it takes about 10 minutes for a 5mL package and about 20 minutes for a 50mL package.

Mixing: Gently invert the solution 5 times before use to ensure uniform mixing.

Addition of Triton X-100 : Pipette the required volume of reagent for the experiment and add an appropriate volume of Triton X-100 to achieve a final concentration of 0.2%. This mixture serves as the detection working solution (for example, add 0.2mL of Triton X-100 to 8mL of ATP detection reagent).

 

II. Detection Procedure

 

1. Cell Culture

 

Use a 96-well plate suitable for chemiluminescence detection, and seed 100μL of cells per well (determine the initial cell density based on the culture time, ensuring that the cell number per well does not exceed 100,000 at the time of detection). Also, set up wells with culture medium without cells as negative controls. Additionally, a cell concentration gradient can be established to obtain optimal experimental results. Culture the cells at 37℃, 5% CO₂, and treat the cells with drugs at appropriate times as needed.

 

2. (Optional) Preparation of ATP Standard Curve

 

Dilute the prepared ATP standard solution with PBS to appropriate concentration gradients, and add 100μL of the standards to each well of the 96-well plate.

 

3. Cell Viability Detection

 

(1) Thaw the frozen luminescent detection reagent and equilibrate it to room temperature (or balance in a 22℃ constant temperature water bath), then take the required volume of detection reagent for the experiment and add an appropriate amount of Triton X-100 (to achieve a final concentration of 0.2%);

(2) Remove the cell culture plate and equilibrate it at room temperature for 10 minutes (or balance at 22℃, with the time not exceeding 30 minutes to avoid prolonged exposure);

(3) Add 100μL of detection reagent to each well of the 96-well plate (due to edge effects in the wells, which may lead to unstable luminescent signals, plating on the edges is not recommended);

(4) Agitate at room temperature for 2 minutes to facilitate cell lysis;

(5) Incubate at room temperature for 10 minutes to stabilize the luminescent signal;

(6) Conduct chemiluminescence detection using a multimode microplate reader. Set the parameters according to the instrument's requirements, with the detection time per well generally being 0.25-1 second, which may need to be adjusted appropriately based on the instrument's detection sensitivity;

(7) Calculate the relative cell viability based on the chemiluminescence readings, or determine the ATP content using the ATP standard curve to derive the relative cell viability.

Note: The detection effect may vary depending on the cell type. For some cells with particularly high ATP content, the chemiluminescence readings may continue to rise when the cell number exceeds 100,000, but the linear relationship may be lost.

 

III. Precautions

 

1. Temperature: The reagent contains luciferase, and repeated freeze-thaw cycles can affect its activity. It is recommended to aliquot and store the reagent at -20℃ in the dark. Both the reagent and cell samples should be equilibrated to room temperature before use to avoid the impact on enzyme catalysis;

2. Chemical Factors: The reaction rate and luminescence intensity of luciferase are influenced by the chemical environment. There are differences in luminescence intensity and decay rates among different types of culture media and sera. Additionally, high drug concentrations may interfere with the luciferase reaction, affecting the luminescence signal. It is advisable to set up control wells with cell culture medium containing the drug to exclude solvent interference. If the culture medium significantly affects luminescence intensity, it can be removed before detection and rinsed once with PBS. In this case, the amount of Triton X-100 added should be halved to a final concentration of 0.1%;

3. Light Sensitivity: The reagent is sensitive to light, and exposure to light during storage can accelerate the decay of luminescence intensity. If transferring the reagent to another container, ensure it is stored in the dark;

4. ATP Contamination: It is recommended to wear masks and latex gloves during operation to avoid contact with surfaces and equipment that may be contaminated. Avoid repeatedly inserting pipette tips into the reagent bottle during the procedure.

 

Recommended Products

Catalog No.	Product Name	Specification
abs50065	2D Luminescent Cell Viability Assay	50mL×2
abs50059	2D/3D/Organoid ATP Viability Assay Kit	100mL

 

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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