Cell Function FAQs

It generally ranges between 0.1 and 0.2.

These two kits differ in their detection principles. BrdU primarily relies on immunological detection, while EdU utilizes a chemical reaction known as click chemistry. Due to its advantages such as simple operation, high sensitivity, minimal damage to cells, and good compatibility with multiplex staining, EdU has gradually become a replacement for BrdU and a new standard for cell proliferation detection.

EdU should be directly added to the culture medium of the adherent cells for incubation. The incubation time should be determined based on the cell doubling time.

The click reaction solution volume for a 12-well plate is 400 μL. For other plate formats, please refer to Appendix Table 1 in the manual.

It is not recommended to do so. Low-concentration EdU should be freshly prepared before use. The 10 mM EdU stock solution should be aliquoted upon first use. It is recommended to aliquot into 50 μL per vial or into smaller volumes as needed for experiments, and store at -20°C.

The 50 Assays kit contains 200 μL of EdU (10 mM), which can be prepared to a concentration of 50 μM in 40 mL. If using a 6-well plate with 2 mL per well, it would be exactly enough for 20 samples. It is recommended to use a 24-well plate with 500 μL per well, as the concentration and incubation time need to be confirmed through preliminary experiments, which will consume some reagents.

For a 24‑well plate, round coverslips with a diameter of 14 mm or square coverslips measuring 10 mm × 10 mm are typically used. The commonly used glass slide size is 75 mm × 25 mm; its width matches the edge of the plate for easy handling, and its length is sufficient to accommodate multiple coverslips for batch staining or observation.

Multi-well plates can be used for incubation; 96, 48, 24, 12, and 6-well plates are all suitable, with different liquid volumes used for different well plates.

The incubation after termination is to allow cells to expel unbound free CFSE dye, thereby reducing background fluorescence and improving labeling uniformity. This step cannot be omitted.

The detection method uses a substrate containing a highly selective caspase-4 recognition sequence (LEVD). Each caspase enzyme has its own preferred cleavage sequence. The LEVD sequence is the classic and most preferred cleavage site for caspase-4. Even when using an apoptosis inducer like Staurosporine, which activates multiple caspases, the fluorescent signal generated in this assay originates entirely from caspase-4 activity. The reasons are as follows: 1.The substrate remains non-fluorescent until it is cleaved. 2.Only activated caspase-4 can recognize and cleave the LEVD sequence. 3.This cleavage releases a DNA-binding dye, which then enters the nucleus, binds to DNA, and produces a strong green fluorescence signal. 4.Other caspases activated by Staurosporine (such as caspase-3 and -8) have distinctly different recognition sequences (e.g., DEVD or IETD) and therefore cannot cleave the LEVD sequence. As a result, they cannot generate any signal in this assay.

The molar concentration of the provided EdU in the kit is 10 mM, with a molecular weight of 252.23 g/mol. It is calculated that the EdU concentration is 2.5 mg/mL.