LiveONLY™ Nuclear Green and LiveONLY™ Nuclear Red are fluorescent nuclear stains specifically developed for exclusive labeling of viable cells, exhibiting no detectable signal in dead or fixed cells. These cell-permeant dyes selectively accumulate in the nuclei of live cells without the need for fixation, permeabilization, or wash steps. Unlike conventional DNA-binding dyes that stain both live and dead cells, LiveONLY™ dyes generate fluorescence only in metabolically active, membrane-intact cells, enabling high-contrast nuclear labeling in live-cell populations (Figure 1). The dyes emit in green and red channels, respectively, and are fully compatible with standard filter sets, facilitating multiplexing in fluorescence-based assays. Their rapid staining kinetics, low background, and robust fluorescence performance make them well-suited for real-time viability assessment, live-cell tracking, and high-content screening applications.

Accurate nuclear staining is essential in live-cell imaging applications, including cell tracking, viability analysis, and high-content screening. However, many fluorescent DNA-binding dyes used in live-cell assays produce nuclear signal in both viable and non-viable cells, often persisting even after membrane compromise or cell death. This nonspecific nuclear labeling introduces background signals from dead cells, leading to data misinterpretation and reducing assay sensitivity in applications that depend on clean segmentation of live-cell populations (1,2).

In dynamic assays where cell viability is changing over time, such as cytotoxicity testing or kinetic drug response studies, the presence of nuclear signal in dead cells leads to false positives and reduces confidence in nuclear-based quantification. These issues are further amplified in no-wash protocols or high-throughput screens where removal of excess dye is not feasible (3,4).

To address this limitation, LiveONLY™ Nuclear Green and LiveONLY™ Nuclear Red were developed as selective live-cell nuclear dyes that generate signal exclusively in viable cells. These dyes provide clear nuclear labeling without fluorescence in fixed or membrane-compromised cells, reducing background interference and enhancing data clarity. Their live-cell exclusivity eliminates the need for counterstains or viability gates, supporting robust imaging performance across diverse assay formats.

HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution (100 U/mL penicillin and 100 µg/mL streptomycin). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Cells were incubated with 1X working solution of either LiveONLY™ Nuclear Red (Cat. No. 17688, AAT Bioquest) or LiveONLY™ Nuclear Green (Cat. No. 17687, AAT Bioquest) prepared in HHBS (Cat. No. 20011, AAT Bioquest). The staining solution was added directly to the wells and incubated for 15-30 minutes at 37 °C, protected from light. The cells were co-stained with spectrally distinct mitochondrial stains, MitoDNA™ Green 530 (Cat. No. 22685, AAT Bioquest) or CytoFix Red Mitochondrial Stain (Cat. No. 23200, AAT Bioquest). The experiment was performed on live cells and 4% PFA fixed cells.

Imaging was performed using a Keyence BZ-X fluorescence microscope equipped with standard filter sets. FITC and TRITC filters were used to detect LiveONLY™ Nuclear Green and LiveONLY™ Nuclear Red, respectively. For mitochondrial co-staining, MitoDNA™ Green 530 was imaged using a GFP/FITC filter, while Cytofix Red Mitochondrial Stain was imaged using the Cy3/TRITC channel. Imaging was conducted on both live and 4% paraformaldehyde (PFA)-fixed cells under identical acquisition settings. Exposure parameters were kept consistent across conditions to allow direct comparison of signal specificity and nuclear selectivity.

Staining with LiveONLY™ Nuclear Green in the presence of a mitochondrial counterstain resulted in bright, selective nuclear fluorescence in live cells. Fixed or dead cells showed strong mitochondrial staining but no nuclear signal, confirming the exclusive activity of the LiveONLY™ dye in viable cells (Figure 2). The green nuclear fluorescence was stable and well-separated from the mitochondrial channel when imaged using FITC filter settings.

In cells co-stained with LiveONLY™ Nuclear Red and a mitochondrial marker, the red nuclear fluorescence was confined to live nuclei, while the mitochondrial signal remained cytoplasmic and intact across all cells. As observed with the green dye, nuclear staining was absent in dead or fixed cells, even when mitochondrial labeling persisted. The red nuclear signal was clearly distinguishable using standard TRITC filters (Figure 3). These results support the dye's suitability for multiplexed live-cell assays where clean separation of organelle-specific signals is required.

The selective staining capability of LiveONLY™ Nuclear Red and LiveONLY™ Nuclear Green enables accurate nuclear visualization exclusively in viable cells, addressing a common limitation observed with many DNA-binding dyes that persist in dead or fixed cells. The absence of nuclear fluorescence in membrane-compromised or PFA-fixed samples demonstrates the dyes' suitability for viability-dependent cellular assays, where distinguishing live from dead populations is crucial.

Co-staining with mitochondrial markers confirmed the spatial separation of nuclear and cytoplasmic compartments. The ability to perform clean, multiplexed imaging without wash steps or viability gating simplifies assay workflows and reduces the need for additional viability dyes or image-based exclusion strategies. These properties make LiveONLY™ dyes especially valuable for real-time cell tracking, cytotoxicity screening, and high-content phenotypic assays. Their compatibility with standard filter sets and easy protocol under live-cell conditions supports seamless integration into both manual and automated imaging platforms. LiveONLY™ Nuclear Red and Green offer a reliable and convenient solution for live-cell nuclear staining with high specificity and minimal background interference.