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Cat. No.: CVAKR-500 (for 500T)

Cat. No.: CVAKR-2500 (for 2500T)

Cat. No.: CVAKR-10k (for 10000T)

Description

Cell Viability Assay Kit with Resazurin, abbreviated CTF Cell Viability Assay Kit or CTF, and also known as the Alamar Blue Cell Viability Assay Kit, is a rapid and highly sensitive assay based on the principle that dehydrogenases in viable cells reduce non‑fluorescent Resazurin into strongly red‑fluorescent Resorufin. This kit is suitable for both adherent and suspension cells and can be applied to proliferation and cytotoxicity assays for most cell types, typically using fluorescence microplate readers.

Resazurin—also known as sodium resazurin, resazurin dye, azoresorcinol, Alamar Blue, etc.—is a deep red crystalline compound with a greenish sheen. It is cell‑permeable, non‑toxic, and exhibits weak blue fluorescence. As a redox indicator, it changes color according to the cellular redox state. It is also used as a pH indicator, with a color transition from orange at pH 3.8 to deep purple at pH 6.5.

In its oxidized form, Resazurin appears blue‑purple and is nearly non‑fluorescent. After entering cells, it is reduced by metabolic reducing equivalents NADH and NADPH under the catalysis of dehydrogenases, producing Resorufin, which is pink and strongly fluorescent. The fluorescence intensity generated in this process is proportional to the number of viable cells, reflecting overall cell activity.

Common fluorescence detection wavelengths for Resorufin are:

• Excitation: ~560 nm

• Emission: ~590 nm

Features

• The kit offers high sensitivity and a wide linear detection range. In a 96‑well plate, the minimum detectable cell number is approximately 100 cells per well. With appropriate incubation times, most cell types show a good linear correlation within 100–50,000 cells, while some cell types maintain linearity up to 100,000 cells. The upper limit of linear detection varies significantly among different cell lines. For example, HeLa cells exhibit good linearity up to 50,000 cells, whereas L‑929 and MOLM‑13 cells maintain linearity up to 100,000 cells. For most cell types, fluorescence continues to increase even after exceeding 50,000–100,000 cells, although the response may no longer be linear. For a minority of cell types, fluorescence may slightly decrease at very high cell densities.
• The kit is extremely easy to use. It contains a single tube of ready‑to‑use 10× detection reagent, requiring no additional preparation. All assay steps are performed directly in the same cell‑culture plate. Simply add the detection reagent to the wells, incubate briefly, and measure fluorescence. No washing, cell harvesting, medium removal, or solubilization steps are required. The reduced product, resorufin, is highly water‑soluble, eliminating the need for extra processing.
• The assay is highly convenient. The detection reagent is non‑toxic to cells, allowing repeated fluorescence readings at different time points to determine the optimal measurement window. For most cell types, ideal results are obtained after 1–4 hours of incubation. Longer incubation enhances the signal from low cell numbers, improving sensitivity, but high‑density samples may reach a plateau and lose linearity. For example, in a 96‑well plate, 50,000 HeLa cells typically yield optimal results after 1 hour, whereas MOLM‑13 cells may require 4–6 hours for optimal signal.
• The kit exhibits excellent stability and high safety. The detection reagent remains effective after 10 freeze–thaw cycles and after storage at 4 °C or room temperature for 7 days. It does not interfere with the electron transport chain and has minimal impact on cell viability. It is non‑toxic to users and environmentally safe.
• The kit is highly compatible with downstream applications. Because it does not damage cells, the same cells can be used for subsequent experiments such as Western blotting, qPCR, or enzyme activity assays.
• The kit is widely applicable and flexible. It supports both adherent and suspension cells and is suitable for nearly all cell types. It is ideal for small‑scale assays as well as high‑throughput screening (HTS).
• For a 96‑well plate, using 10 µL reagent per 100 µL cell suspension, each 1 mL of reagent supports 100 assays. For a 384‑well plate, using 2.5 µL reagent per 25 µL cell suspension, each 1 mL supports 400 assays.

Storage

• Store at –20 °C, protected from light; stable for one year.
• Store at 4 °C, protected from light; stable for one month.

Precautions

• Long-term exposure to light increases background fluorescence during detection, so the reagent should be protected from light at all times.
• The product tolerates up to five freeze–thaw cycles without significant loss of performance. However, for first-time use, it is recommended to mix thoroughly, aliquot appropriately, and store at –20 °C protected from light to minimize repeated freeze–thawing. For frequent short-term use, storage at 4 °C protected from light is convenient.
• Fluorescence measurements should be performed using black 96‑well or 384‑well plates. Using standard clear plates may cause signal crosstalk between adjacent wells.
• Different cell types reduce Resazurin to Resorufin at different efficiencies depending on metabolic activity and incubation time. Most cell types achieve optimal detection within 1–4 hours, but the required incubation time may vary. A preliminary experiment is recommended to determine the appropriate cell number and optimal incubation duration for the specific cell line being used.
• The kit can also be used for absorbance detection at 570 nm (with 600 nm as the reference wavelength), but fluorescence detection is preferred due to its higher sensitivity.
• This product is intended for research use only. It is not for clinical diagnosis or therapy, not for use in food or drug applications, and should not be stored in residential environments.
• For safety, wear a lab coat and disposable gloves during handling.