In biological experiments such as pharmacological screening, tumor proliferation research, and in vitro cytotoxicity evaluation, accurate live cell counting is the core prerequisite for reliable data. Traditional metabolism-dependent detection reagents like MTT and CCK-8 are susceptible to metabolic fluctuations and drug interference, often resulting in deviations. Conventional nucleic acid staining-based kits are frequently affected by extracellular free DNA interference, with non-specific fluorescence elevating background values, hindering high-throughput screening applications. U-ABio's self-developed CyQuantiFluor™ Cell Viability Detection Kit leverages dual-dye collaborative shielding technology, bypassing metabolic dependence limitations and excelling with its homogeneous operation, making it the preferred tool for cell proliferation, cytotoxicity, and small-molecule drug high-throughput screening.

The kit's core consists of a DNA-specific binding dye paired with a background shielding dye, which work in tandem to achieve exclusive live cell labeling. The DNA-binding dye exhibits extremely low fluorescence in its free state, penetrates intact live cell membranes, and binds to nuclear DNA, significantly enhancing fluorescence. The fluorescence intensity correlates linearly with intracellular DNA content, enabling live cell quantification. The accompanying background shielding dye cannot penetrate intact live cell membranes but quenches interference signals from dead cell-released free DNA, system impurity DNA, and dye-bound fluorescence, ensuring the captured fluorescence originates solely from live cell nucleic acid complexes. Unlike dehydrogenase activity-dependent colorimetric reagents, CyQuantiFluor™ directly quantifies live cell genomic DNA content, unaffected by cellular metabolic states or drug-induced metabolic inhibition, delivering significantly higher accuracy than traditional enzyme-based systems.

The product features a fully homogeneous formulation, enabling one-step detection without media changes, washing, or reaction termination. Simply add an equal volume of 2× working solution to cell culture medium, minimizing pipetting errors from multi-step operations and adapting to high-throughput 96-well or 384-well plate formats. With high signal-to-noise ratio, excellent batch consistency, and outstanding solution stability, the kit is compatible with various adherent and suspension cell models. It is ideal for routine cell proliferation kinetics, exogenous compound cytotoxicity assessment, and particularly excels in high-throughput screening of antitumor drug candidates and tumor cell proliferation inhibition studies.

Use black-walled, clear-bottom 96-well plates, seeding 100 μL of cells at an appropriate density per well;

Treat cells with test compounds for proliferation/toxicity models and incubate to establish the model;

Prepare working solution: Dilute 250× DNA-binding dye and 50× background shielding dye in PBS, HBSS, or complete cell culture medium to prepare 2× detection solution. For a 12 mL system, add 48 μL nucleic acid dye and 240 μL shielding dye to 11.7 mL HBSS, vortex thoroughly;

Add 100 μL of 2× detection reagent to each well containing 100 μL cell suspension, maintaining a strict 1:1 ratio;

Incubate at 37°C, 5% CO₂ in the dark for 1–6 h (extend incubation to enhance sensitivity; optimize duration via pilot experiments). For suspension cells, centrifuge at low speed to settle cells before reading;

Read fluorescence using a microplate reader in bottom mode (FITC channel: Ex 490 nm / Em 520 nm).

Absolute fluorescence values vary across plates due to cell type and drug properties. Include solvent blank controls and positive toxicity controls, normalizing all sample data to controls before calculating cell viability or proliferation inhibition rates to ensure cross-group comparability.

From basic cell research to pharmaceutical high-throughput compound screening, CyQuantiFluor™ addresses traditional cell viability detection shortcomings with its high specificity, ease of use, and anti-interference properties, delivering a stable and reliable nucleic acid quantification solution for in vitro pharmacological studies.