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Cat. No.: CVAKG-500 (for 500T)

Cat. No.: CVAKG-2500 (for 2500T)

Description

Cell Viability Assay Kit with GF‑AFC, abbreviated CTF Cell Viability Assay Kit or CTF, and also known as the GF‑AFC Cell Viability Assay Kit, is a rapid and highly sensitive assay based on the principle that proteases in viable cells cleave the non‑fluorescent substrate GF‑AFC to release the strongly blue‑fluorescent product AFC. This kit is suitable for both adherent and suspension cells and can be used for proliferation assays and cytotoxicity testing across a wide range of cell types, typically in fluorescence microplate‑reader systems.

GF‑AFC, also known as Gly‑Phe‑AFC (Glycyl‑Phenylalanyl‑7‑Amino‑4‑Trifluoromethylcoumarin), has the chemical formula C₂₁H₁₈N₃O₄F₃ and a molecular weight of 433.38. It is a cell‑permeable protease substrate that can be hydrolyzed by intracellular proteases. Upon cleavage, it releases a fluorescent moiety that can be used to assess cell viability, cell proliferation, or cytotoxicity. GF‑AFC is also widely used to evaluate the enzymatic activity of cathepsins B and L, and for screening inhibitors or activators of these proteases.

GF‑AFC itself is nearly non‑fluorescent. After entering viable cells, it is cleaved by intracellular proteases to generate AFC (7‑Amino‑4‑Trifluoromethylcoumarin). AFC exhibits a strong blue fluorescence, and the fluorescence intensity is proportional to the number of viable cells, reflecting overall cellular activity.

Common fluorescence detection wavelengths for AFC are:

• Excitation: ~380 nm

• Emission: ~490 nm

Features

• The kit provides high sensitivity and a broad linear detection range. In a 96‑well plate, the minimum detectable cell number is about 100 cells per well. With appropriate incubation times, most cell types show a good linear correlation within 100–20,000 cells, while some cell types maintain linearity up to 100,000 cells. The upper limit of linear detection varies substantially among different cell lines. For example, HeLa and MOLM‑13 cells exhibit good linearity up to 20,000 cells, whereas Jurkat cells maintain linearity up to 100,000 cells. For most cell types, the response may become non‑linear once cell numbers exceed 20,000–50,000, although fluorescence intensity typically continues to increase.
• The kit is very easy to use. The GF‑AFC detection reagent (1000×) only needs to be diluted 1:1000 with Assay Buffer to prepare the GF‑AFC working solution, which is then added to the cell‑culture plate at an equal volume to the medium already present in each well. After a brief incubation, fluorescence can be measured directly. All assay steps are completed within the same culture plate, with no washing, no cell harvesting, and no medium removal required. The cleavage product AFC is highly water‑soluble, so no additional solubilization steps are necessary.
• The kit is highly convenient to use. The GF‑AFC working solution is non‑toxic to cells, allowing fluorescence readings to be taken repeatedly at different time points, which provides flexibility in determining the optimal measurement window. For most cell types, an incubation period of approximately 1–3 hours yields ideal detection performance. Extending the incubation time significantly enhances the signal from wells containing low cell numbers, thereby improving sensitivity, whereas wells with high cell numbers gradually reach a plateau and no longer maintain linearity. For example, in a 96‑well plate, 100,000 HeLa cells per well typically produce an optimal signal after 1 hour of incubation, while MOLM‑13, L‑929, and Jurkat cells generally require 2–3 hours to achieve stronger and more reliable fluorescence signals.
• The kit shows strong correlation with cell‑viability assays based on the Resazurin/Resorufin method. This kit measures viable‑cell number by detecting AFC, the fluorescent product released when intracellular proteases cleave GF‑AFC. In contrast, the Cell Viability Assay Kit with Resazurin measures cellular metabolic activity, in which intracellular dehydrogenases reduce Resazurin to the fluorescent product Resorufin. Although the two assays rely on independent biochemical mechanisms, both provide efficient and sensitive measurements of cell viability.

Storage

• Store at –20 °C; stable for one year.
• The GF‑AFC detection reagent (1000×) must be stored protected from light.

Precautions

• Prolonged exposure of the 1000× GF‑AFC detection reagent to light may increase background fluorescence during measurement and should be avoided.
• The 1000× GF‑AFC detection reagent tolerates up to 10 freeze–thaw cycles without significant loss of performance, but for first‑time use it is recommended to aliquot and store at –20 °C protected from light to minimize repeated freeze–thawing.
• Fluorescence measurements should be performed using black 96‑well or 384‑well plates. Using standard clear plates may cause signal interference between adjacent wells.
• The efficiency with which different cell types cleave GF‑AFC into AFC depends on the protease activity of the cell line and the incubation time with the GF‑AFC working solution. For most cell types, an incubation period of 1–3 hours yields optimal results, though the required time may vary. A preliminary experiment is recommended to determine the appropriate cell number and optimal incubation time for the specific cell type.
• The 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.