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An integrated live cell monitoring

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Application Note: An integrated live cell monitoring system for stable imaging of suspension cells in immunological research

Using Celloger Mini Plus to observe morphological changes and phagocytic activity in macrophage cell line


This Application Note demonstrates real-time imaging and live cell analysis of LPS-stimulated Raw264.7 macrophage cells using the Celloger Mini Plus to monitor morphological changes and phagocytic activity in suspension-based immunological research. Researchers studying white blood cell function often face instability and focus drift when using traditional microscopes, particularly during suspension cell imaging outside incubator conditions.

To address this challenge, Yamato Scientific America highlights the Celloger integrated live cell monitoring system, which performs stable, multi-position imaging entirely inside a CO₂ incubator. By eliminating stage movement and maintaining environmental control, Celloger enables continuous observation without cell disturbance.

Key results show that LPS stimulation induced macrophage differentiation, increased cell spreading, and enhanced phagocytosis assays with fluorescent latex beads. Real-time bright field and green fluorescence imaging captured dynamic bead uptake and cell division events, while built-in confluency analysis quantified proliferation. This stable imaging platform improves reproducibility, reduces experimental variability, and supports advanced immunology workflows. Download the full application note to explore the detailed protocol and comprehensive data set demonstrating the advantages of Celloger for suspension cell and macrophage research.

As white blood cells responsible for immune function are suspension cells that travel along blood vessels, immunology studies often use various suspension cell lines originating from white blood cells. Dealing with suspension cells, unlike adherent cells, slight movement of a plate when locating it on the microscope causes the cells to float. Aside from the problems caused by temperature and CO2 instability, it is in fact not possible to use a traditional microscope to monitor cells in real time. Therefore, in order to stably monitor suspension cells, a live cell imaging device such as Celloger that operates inside an incubator is essential. In addition, with Celloger Mini Plus, the camera inside the system moves to capture the images of cells in multiple positions to keep the cell sample in a steady state instead of having a movable stage with a plate on it. When the suspension cells were monitored both by Celloger and microscope, imaging with Celloger was more stable compared to using a microscope in which several cells were out of focus (Figure 1).

For suspension cell imaging using an inverted microscope, cell fluctuation is inevitable since the sample must be taken out of an incubator, placed on the microscope stage, and be moved to locate other positions within the plate. In this process, many cells are observed to be floating off the floor and are out of focus (Left image). On the other hand, imaging with Celloger Mini Plus enables the entire process of imaging inside an incubator. The plate is stably fixed on the device and multiple positions within the plate can be imaged by moving the camera inside the system; thus nothing causes cell floating and no cells are observed to be out of focus (Right image). 

White blood cells as a part of the immune system fight infection and defend the body against foreign materials. A first line of defense system known as innate immunity generates fast inflammatory responses to immediately prevent the spread and movement of foreign pathogens throughout the body. Because activation of the innate immune system is initiated within hours and generates rapid inflammatory responses, it is important to monitor the various cellular defenses occurring in this process in real time. An important function of innate immunity is the rapid recruitment of immune cells to an infected area. Among white blood cells, monocytes infiltrate tissues and differentiate into macrophages, while inducing an immune response against invading pathogens through phagocytosis. We performed live cell imaging with Celloger Mini Plus (Bright field & green fluorescence channels, 10X objective) using the Raw264.7 cell line, which represents the functional characteristics of macrophage and is well known for changes caused by lipopolysaccharide (LPS).

When Raw264.7 cells were stimulated by LPS, the differentiated cells increased. As round and thick cuboidal cells in a loosely adhered state were differentiated, they slowly spread and adhered more firmly in a spindle form, and this was observed through images taken every 15 minutes by Celloger Mini Plus (10X optics).​

According to the study by Saxena et al. (2003), Raw264.7 cells stimulated by LPS differentiate into dendritic-like cells. We confirmed through real-time monitoring using Celloger Mini Plus that the cells adhered more firmly and became wider and flatter than the cells without LPS treatment (Figure 2).

This change in the morphology was more visible 11 hours after LPS treatment and lasted for up to 22 hours. Also, LPS stimulation resulted in cell proliferation, which was quantified through Celloger’s confluency analysis function (Figure 3).

By using the time-lapse images (captured via Celloger Mini Plus, 10X optics) and analyzing the cell confluency with the Celloger Mini Plus analysis app, a cell growth graph can be obtained.​

There is a report that LPS-induced activation of macrophage increases phagocytosis through the toll-like receptor 4-dependent pathway. To confirm this in real time, we performed fluorescence imaging using fluorescent latex beads that were engulfed by macrophages. The 2um-sized fluorescence beads swayed easily with the slightest movement and floated above the plate. This not only decreases the bead uptake efficiency of cells but can also make imaging difficult. In fact, even commercially available phagocytosis assay kits recommend only counting bead-engulfed cells by fluorescence microscopy or flow cytometry after properly washing at the endpoint to remove non-engulfed beads. In this experiment, Celloger's real-time imaging showed the entire bead phagocytosis process of the cells and the bead engulfment taking place only for the activated cells that were spread flat with LPS stimulation (Figure 4A).

The way these activated cells behave are expected to be possible because they efficiently move toward the beads in a spindle shape that cause directional migration compared to the round and cube-like shape before differentiation. It was also observed in real time that the beads were divided into daughter cells along with the cytoplasm when the cells were divided after bead uptake (Figure 4B).

   A.  It was observed that the activated Raw264.7 cells after LPS stimulation engulfed the fluorescent bead. Time-lapse images were taken at a 15-minutes interval to observe cells migrate toward the bead and uptake the bead.   

   B.  The engulfed beads inside the cells are divided into daughter cells along with the cytoplasm during mitosis.

In addition, it is possible to obtain dynamic process information for various molecular environments by using the unique characteristics of the fluorescent probe. If a fluorescent dye can be additionally imaged along with the bright field imaging, it can be used for further in-depth studies. Using cell-impermeant nucleic acid stain, cytotoxicity can be evaluated through the increase in the permeability of the dye due to membrane damage during apoptosis. And in the case of neutrophil in particular, NET formation can also be detected by nucleic acid staining. Furthermore, it is possible to quantify the generation of reactive oxygen species in cells using dyes that react with reactive oxygen species or observe the intracellular acidification in the process of endocytosis and phagocytosis using pH-sensitive dye.

Live cell imaging with Celloger Mini Plus enables obtaining high-resolution images of cells while eliminating physical disturbances such as cell damage or shaking. It is an automated system that functions perfectly inside an incubator, eradicating the need to move the device in and out of the incubator. Unlike other devices, Celloger Mini Plus doesn’t have a movable stage but instead, the camera inside the system moves to capture the images of cells in multiple positions. Since the vessel and cell samples are in a steady state, this provides a stable environment for the cells to grow and increases the success rate of cell-based research. Various types of culture vessels are compatible with the system, and it has high position reproducibility for stable scanning performance during multi-point imaging; therefore, Celloger Mini Plus will bring reliable results in immunology research.

Suspension cells tend to float with even slight plate movement, making them go out of focus during imaging. Traditional microscopes require removing the plate from the incubator, causing temperature and CO₂ instability and physical disturbance. This makes stable, real-time monitoring of suspension cells challenging.

Celloger Mini Plus operates entirely inside an incubator, maintaining stable temperature and CO₂ conditions. Instead of moving the plate, the internal camera moves to capture multiple positions, keeping the sample steady. This design prevents cell floating and ensures stable, in-focus images during long-term monitoring.

Yes, Celloger Mini Plus enables real-time monitoring of Raw264.7 macrophage cells stimulated with LPS. Morphological changes—such as cells spreading, flattening, and adhering more firmly—were captured every 15 minutes using bright field imaging. These changes became more visible around 11 hours after LPS treatment and persisted up to 22 hours.

The study used a 10X objective with both bright field and green fluorescence channels. Time-lapse images were taken at 15-minute intervals. This setup allowed researchers to observe both morphological changes and phagocytic activity in real time.

Cell proliferation is quantified using the confluency analysis function within the Celloger Mini Plus analysis app. Time-lapse images are analyzed to generate a cell growth curve. This provides quantitative data on proliferation following treatments such as LPS stimulation.

Yes, Celloger enables real-time visualization of phagocytosis using fluorescent latex beads. In this study, activated Raw264.7 cells engulfed 2 µm fluorescent beads, and the entire uptake process was recorded at 15-minute intervals. This approach allows continuous monitoring instead of relying only on endpoint assays.  

Traditional phagocytosis assays often require washing steps and endpoint fluorescence microscopy or flow cytometry to count engulfed beads. Celloger allows continuous, live imaging of bead uptake without disturbing the cells. This provides dynamic insights into migration, engulfment, and even bead distribution during cell division.

Yes, by combining bright field imaging with fluorescent probes, Celloger Mini Plus can assess cytotoxicity and apoptosis. For example, cell-impermeant nucleic acid stains can detect membrane damage, while other dyes can measure reactive oxygen species or intracellular acidification. This enables deeper analysis of molecular and cellular responses in real time.

The system is suitable for suspension cells, including white blood cell–derived lines such as Raw264.7 macrophages. It supports stable imaging of immune cells involved in innate immunity studies. Additionally, various types of culture vessels are compatible with the device for flexible experimental design.

Celloger Mini Plus functions entirely inside an incubator, eliminating environmental fluctuations and physical disturbances. Its fixed plate design with a moving internal camera ensures high position reproducibility and stable multi-point imaging. This increases experimental reliability and improves the success rate of cell-based immunology research.  


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to access the complete experimental protocol, quantitative imaging data, and detailed analysis of LPS-stimulated Raw264.7 macrophage differentiation and phagocytic activity 

using the Celloger Mini Plus live cell monitoring system.

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Observation of Mitosis