Cell Counting Kit-8 (CCK-8) allows for very convenient assays using highly water-soluble tetrazolium salt. WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H- tetrazolium, monosodium salt] produces a water-soluble formazan dye upon reduction in the presence of an electron mediator. CCK-8 is a one-bottle solution; no premixing of components is required. As a non-radioactive option, CCK-8 enables sensitive colorimetric assays to determine the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is reduced by dehydrogenases in cells, yielding an orange-colored product (formazan), which is soluble in the tissue culture medium. The amount of formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells.

The kit is designed for the spectrophotometric measurement of cell growth based on mitochondrial activity in living cells. MTT measures the activity of living cells through mitochondrial dehydrogenase activity, offering a simple, accurate, and reproducible method. Dehydrogenases in mitochondria reduce MTT to a crystalline purple product, Formazan. These crystals can be dissolved in a specific solvent for measurement. Use a Microplate Absorbance Reader to measure absorbance at a wavelength near 490 nm. As cell numbers increase, more MTT Formazan is formed, leading to a corresponding increase in absorbance.
For best results, cell numbers should be determined during log growth stage. Each test should include a blank containing complete culture medium without cells.

JC-10 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm). Mitochondrial depolarization is reflected by a decrease in the red/green fluorescence intensity ratio. This potential-sensitive color shift is due to the concentration-dependent formation of red fluorescent J-aggregates.
JC-10 can be used as an indicator of mitochondrial potential in various cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. The use of fluorescence ratio detection allows researchers to make comparative measurements of membrane potential and determine the percentage of mitochondria within a population that respond to an applied stimulus, thus revealing subtle heterogeneity in cellular responses. JC-10 is most commonly used to detect mitochondrial depolarization in the early stages of apoptosis.

Calcein-AM is a cell-staining reagent used for fluorescent labeling of living cells, emitting green fluorescence (Ex = 490 nm, Em = 515 nm). The addition of an acetomethoxymethyl ester (AM) group to traditional Calcein increases its hydrophobicity, allowing it to easily penetrate the membrane of living cells. Once inside the cell, Calcein - AM (which is not fluorescent on its own) is cleaved by intracellular esterases to form the nonpermeable polar molecule Calcein, which becomes trapped inside the cell and emits strong green fluorescence. Compared to other similar reagents like BCECF - AM and CFDA, Calcein-AM is the most suitable fluorescent probe for live cell staining due to its very low cytotoxicity and its lack of inhibition on cellular functions such as proliferation and lymphocyte chemotaxis.

Because dead cells lack esterase, Calcein - AM is used only for cell viability testing and short - term labeling of living cells. Therefore, Calcein - AM is often used in combination with dead cell fluorescent probes such as propidium iodide (PI) for simultaneous fluorescent double staining of live and dead cells. Propidium iodide (PI) can not pass through the cell membrane of living cells, but can only pass through the disordered region of the dead cell membrane to reach the nucleus and embed into the DNA double helix of the cell to produce red fluorescence (Ex=535 nm, Em=617 nm). Therefore, PI only stains dead cells. Since both Calcein and PI - DNA can be excited by 490 nm, both live and dead cells can be visualized by fluorescence microscopy. However, only dead cells could be observed with 545 nm excitation. The working principle of this kit is to double dye Calcein - AM and PI to double stain and label live and dead cells, so as to analyze the level of live and dead cells. According to our optimized experimental system, 200 μl cell suspension can be stained for 500 times.