Simultaneous visualization of multiple organelles is essential for understanding subcellular organization and dynamic cellular responses. In this study, we describe a streamlined and robust workflow for multiplex fluorescent staining of five key organelles in HeLa cells using five spectrally distinct dyes. Nuclear DNA, mitochondria, endoplasmic reticulum, actin cytoskeleton, and plasma membrane glycoproteins were labeled using organelle-specific stains. The combination of live-cell and post-fixation staining enabled clear spatial resolution of each compartment with minimal spectral overlap. Composite imaging confirmed high signal specificity and reproducibility across all five channels. This multiplex labeling approach provides a practical and adaptable platform for high-content imaging applications, including phenotypic screening, cell painting assays, and subcellular profiling in fixed and live-cell formats.

Advancements in fluorescence microscopy and cell-based assays have significantly improved the understanding of subcellular structures and their dynamic interactions. The ability to simultaneously visualize multiple organelles within a single cell has emerged as an essential tool for studying organelle-specific functions, cellular architecture, and disease-related perturbations (1,2). Multi-organelle imaging enables the comprehensive mapping of cellular compartments, providing a high-content platform for phenotypic screening, drug discovery, and functional genomics (3). Among the key organelles of interest, the nucleus, mitochondria, endoplasmic reticulum (ER), actin cytoskeleton, and plasma membrane glycoproteins play pivotal roles in maintaining cellular homeostasis, signaling, and stress responses. Their coordinated interactions are often implicated in diverse biological processes such as apoptosis, mitophagy, protein trafficking, and cytoskeletal remodeling (4,5).

Here, we describe a simplified multiplex staining workflow using Nuclear Violet™ LCS1, CytoFix™ Red, ER Tracer™ Green, Phalloidin-iFluor^® 633, and iFluor^®-750 WGA to visualize five major organelles in HeLa cells. The optimized dye combination provided clear spatial resolution, minimal spectral overlap, and compatibility with both live and fixed-cell imaging formats. This assay offers a practical tool for multi-channel fluorescence microscopy and high-content phenotypic analysis.

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.

Fluorescence imaging was performed using a Keyence fluorescence microscope equipped with appropriate filter sets:

Sequential image acquisition was performed to minimize spectral overlap. Composite images were generated to visualize co-localization and cellular morphology.

The multiplex staining protocol successfully enabled simultaneous visualization of five key organelles within HeLa cells using spectrally distinct fluorescent probes. Each dye demonstrated high specificity and intensity under the respective fluorescence channels, confirming effective labeling and minimal spectral bleed-through. Nuclear Violet™ LCS1 effectively stained the nuclei of live HeLa cells and the nuclear morphology remained intact throughout the imaging process (Figure 2A). CytoFix™ Red Mitochondrial Stain yielded a strong red fluorescence (Cy3 channel), localizing precisely to the mitochondrial network (Figure 2B). ER Tracer™ Green labeled the endoplasmic reticulum with bright green fluorescence (GFP channel), outlining the perinuclear and peripheral ER network (Figure 2C). Phalloidin-iFluor^® 633 selectively stained filamentous actin (F-actin), providing a pseudo-yellow signal (Cy5 channel) that highlighted the F-actin structures, indicating preserved cytoskeletal integrity post-fixation (Figure 2D). iFluor^®-750 WGA selectively labeled cell surface glycoproteins with a far-red signal shown in pseudo-violet (Cy7 channel) (Figure 2E). The membrane outlines were clearly visible, providing contrast for cell boundaries.

Overlay of all five fluorescence channels resulted in a high-quality composite image showing distinct and non-overlapping signals for each organelle (Figure 3). The multiplexed staining demonstrated clear spatial resolution and compatibility of all dyes within a single imaging session. No significant crosstalk or signal interference was observed between channels. These results confirm the feasibility and robustness of the developed staining workflow using cell organelle staining reagents for simultaneous imaging of nucleus, mitochondria, endoplasmic reticulum, cytoskeleton, and plasma membrane in fixed cells.

The ability to visualize multiple organelles in a single sample with clear spatial separation offers an efficient approach to studying cellular organization. The staining workflow described here makes use of spectrally distinct fluorescent dyes to support multi-channel imaging without adding procedural complexity. The strong compartment-specific signals observed across all five channels confirm that these dyes work well together and do not interfere with one another during acquisition.

This type of multiplex labeling is particularly useful in experiments where organelle morphology or interactions may shift in response to treatment, stress, or disease models. The method is well-suited for high-content imaging applications such as phenotypic screening, cytotoxicity assays, and morphology-based profiling. Since the protocol can be easily adapted or extended with additional markers, it also offers flexibility for broader use in cell biology and drug discovery studies.