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Cat. No.: L3D-100 (for 100T)

Cat. No.: L3D-500 (for 500T)

Cat. No.: L3D-2500 (for 2500T)

Description

Luminescent 3D Cell Viability Assay Kit is a reagent kit used for quantitative detection of overall cell viability in 3D-cultured cells with ultra-high sensitivity and signal stability. It utilizes a chemiluminescent method to measure the intracellular ATP content.

Traditional cell culture is mostly conducted in a two-dimensional (2D) format. However, researchers have discovered significant differences in growth patterns, morphology, cell structure, cell differentiation, and biological functions between cells cultured in 2D and cells under in vivo physiological conditions. These differences can lead to significant discrepancies between experimental results and the actual in vivo situation. Three-dimensional (3D) cell culture techniques provide a better simulation of the microenvironment in which cells survive in the body. Cells cultured in 3D maintain their normal biological functions and better mimic in vivo tissues, accurately reflecting the interactions between cells and between cells and the extracellular matrix. The cellular response to external and internal stimuli is also more similar to the in vivo situation. As a result, 3D cell culture has become a more reliable in vitro cellular model, capable of producing experimental results that are closer to or consistent with in vivo experiments.

3D tumor cell models are increasingly used to understand disease mechanisms and drug development. In 2D cell culture, tumor cells can uniformly obtain nutrients and oxygen through their single-sided cell membrane. In contrast, 3D-cultured tumor cell aggregates have fewer opportunities for internal cells to obtain nutrients and oxygen, resulting in a natural nutrient and oxygen gradient. This better mimics the in vivo microenvironment and allows for more accurate modeling of tumors, making it more suitable for small molecule drug screening or studying tumor-related molecular mechanisms. It also enables more accurate predictions of in vivo responses, efficacy, or toxicity of drug treatments. Additionally, 2D tumor cell models have limitations in terms of the loss of the original tumor's genetic heterogeneity and the selection of dominant clones after passaging, reducing their clinical relevance. Compared to 2D cell models, 3D cell spheroids or organoids often provide more reliable research results, simplifying and accelerating the drug evaluation process. Since the establishment of the first intestinal organoid in 2009, 3D cell and organoid research has expanded to many tissue systems and has become one of the hottest areas in life sciences.

With the increasing maturity of 3D cell culture techniques, they have become widely used research tools and are gaining popularity. Their applications are becoming more extensive in basic research and drug discovery. However, assessing cell viability under 3D culture conditions remains a challenge.

ATP, as the most important energy molecule, plays a crucial role in various cellular physiological and pathological processes. ATP is an important indicator of cellular metabolism and a characteristic molecule of metabolically active cells. It shows a good linear relationship with the number of viable cells. Therefore, ATP content can reflect the number of viable cells, and cell viability can be measured by determining ATP content.

The detection reagents in this assay kit have been optimized to effectively penetrate the interior of 3D spheroids formed by cell culture and lyse all cells to release ATP. Cell viability is then determined by measuring ATP content. The principle of detecting cell viability using ATP is shown in Figure 1. Through the ATP-dependent luminescence reaction catalyzed by luciferase, ATP can be quantified by measuring chemiluminescence. Since ATP content reflects the number of viable cells and is proportional to the luminescence intensity, cell viability or cell number can be calculated simply by measuring the chemiluminescence intensity.

Features

• This product is easy to use, provides stable readings, and has a fast detection speed, with the entire assay taking approximately 30 minutes to complete. It offers a simpler and more convenient method compared to other 3D cell viability assays. Simply dissolve the detection substrate (lyophilized powder) provided in the kit using the supplied assay buffer to prepare the luminescent assay reagent. Mix the reagent with the cell culture medium or other appropriate volumes, shake for 5 minutes, and then incubate at room temperature for 25 minutes before performing the chemiluminescent detection. There is no need to wash the cells or replace/remove the culture medium. Additionally, the chemiluminescent signal is relatively stable, with a decrease of no more than 10% within the first 10 minutes after the reaction starts and no more than 30% within 30 minutes. The signal has a long half-life of 2-3 hours.
• This product offers high detection sensitivity and a wide linear range for sample measurements. When using this product to detect the viability of HCT-116 cells cultured in Corning® spheroid microplates for 72 hours, even with a seeding density of only 10 cells per well, the luminescent intensity can reach approximately 5 times that of the blank wells, indicating high detection sensitivity. Regarding the linear relationship between luminescent intensity and cell number, for cells cultured in a 3D environment, factors such as contact inhibition of cell proliferation, decreased metabolic activity or cell death in the central region of large cell spheroids may result in a curve-like relationship between the luminescent signal intensity and the number of cells seeded. However, the luminescent signal intensity tends to increase with an increase in the number of seeded cells.
• This product demonstrates good stability. It can be stored long-term at -20°C, and the prepared luminescent assay reagent for 3D detection shows good stability, with no significant impact on the detection results after five freeze-thaw cycles, and a decrease of no more than 10% after ten freeze-thaw cycles. The luminescent assay reagent for 3D detection remains unaffected when stored at 4°C for three days, with a decrease of no more than 10% after seven days. It retains over 80% of the detection efficiency when stored at room temperature for one day, and maintains over 60% of the detection efficiency when stored at 37°C for one day.
• This product offers flexible and convenient usage. It is suitable for the detection of both small and large sample sizes, making it ideal for high-throughput screening applications. It can be used with various 3D cell culture methods, including cell hanging drop plates, ultra-low attachment cell culture plates, plates coated with matrix or Matrigel, and plates coated with agarose.