# 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** 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 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 essential¹. With **Celloger Mini Plus**, the camera inside the system moves to capture 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 suspension cells were monitored both by **Celloger** and a microscope, imaging with **Celloger** was more stable compared to using a microscope in which several cells were out of focus. \[Image placeholder: Figure 1. Advantages of using Celloger during suspension cell imaging\] For suspension cell imaging using an inverted microscope: 1. The sample must be taken out of an incubator. 2. It is placed on the microscope stage. 3. The stage is moved to locate other positions within the plate. 4. Cells float off the floor and become out of focus. In contrast, imaging with **Celloger Mini Plus**: - Enables the entire imaging process inside an incubator. - Keeps the plate stably fixed on the device. - Moves the internal camera to image multiple positions. - Prevents cell floating and eliminates out-of-focus cells. ------------------------------------------------------------------------ ## Introduction White blood cells, as 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 prevent the spread and movement of foreign pathogens throughout the body. Because activation of innate immunity is initiated within hours and generates rapid inflammatory responses, it is important to monitor various cellular defenses in real time. An important function of innate immunity is the rapid recruitment of immune cells to infected areas. Among white blood cells: - **Monocytes** infiltrate tissues and differentiate into **macrophages**. - Macrophages induce immune responses against invading pathogens through **phagocytosis**. Live cell imaging was performed using **Celloger Mini Plus** (Bright field & green fluorescence channels, **10X objective**) with the **Raw264.7 cell line**, which represents macrophage functional characteristics and is known for changes caused by **lipopolysaccharide (LPS)**². ------------------------------------------------------------------------ ## Results ### Monitoring Morphological Changes with **LPS Stimulation** \[Image placeholder: Figure 2. Monitoring morphological changes in Raw264.7 cells with LPS stimulation\] When **Raw264.7** cells were stimulated by **LPS**, differentiated cells increased. Initially round and thick cuboidal cells in a loosely adhered state slowly spread and adhered more firmly in a spindle form. Images were taken every **15 minutes** using **Celloger Mini Plus (10X optics)**. According to Saxena et al. (2003), Raw264.7 cells stimulated by LPS differentiate into dendritic-like cells³. Real-time monitoring confirmed that: - Cells adhered more firmly. - Cells became wider and flatter compared to untreated cells. Morphological changes became more visible **11 hours after LPS treatment** and lasted up to **22 hours**. ### Confluency Analysis and Proliferation **LPS stimulation** resulted in cell proliferation quantified using **Celloger's confluency analysis function**. \[Image placeholder: Figure 3. Growth curve of LPS-stimulated cells through confluency analysis\] Using time-lapse images captured via **Celloger Mini Plus (10X optics)** and analyzing cell confluency with the Celloger Mini Plus analysis app, a cell growth graph was obtained. ### Real-Time Phagocytosis Monitoring LPS-induced activation of macrophages increases phagocytosis through the **toll-like receptor 4-dependent pathway**²⁴. To confirm this in real time: - Fluorescent imaging was performed using **2 µm fluorescent latex beads**. - Beads easily swayed and floated with slight movement. - Bead uptake efficiency decreases under unstable conditions. - Commercial phagocytosis assay kits recommend endpoint counting after washing non-engulfed beads⁵⁶. **Celloger Mini Plus** enabled real-time observation of: - The entire bead phagocytosis process. - Bead engulfment occurring only in activated cells spread flat with **LPS stimulation**. \[Image placeholder: Figure 4. Phagocytosis of LPS-stimulated Raw264.7 cell observed with Celloger Mini Plus (Bright field & green fluorescence channel, 10X optics)\] #### Figure 4A Activated **Raw264.7** cells after LPS stimulation engulfed fluorescent beads. Time-lapse images were taken at **15-minute intervals** to observe migration toward and uptake of beads. #### Figure 4B Engulfed beads inside cells were divided into daughter cells along with the cytoplasm during **mitosis**. Activated cells efficiently moved toward beads in a spindle shape, enabling directional migration compared to round, cube-like undifferentiated cells. ------------------------------------------------------------------------ ## Advanced Fluorescence Applications Dynamic molecular process information can be obtained using fluorescent probes. When fluorescent dyes are imaged alongside bright field imaging, further in-depth studies are possible. Applications include: - **Cytotoxicity evaluation** using cell-impermeant nucleic acid stains detecting membrane damage during apoptosis⁷. - Detection of **NET formation** in neutrophils via nucleic acid staining⁸. - Quantification of **reactive oxygen species (ROS)** generation using ROS-reactive dyes⁹. - Observation of **intracellular acidification** during endocytosis and phagocytosis using pH-sensitive dyes¹⁰. ------------------------------------------------------------------------ ## Conclusion Live cell imaging with **Celloger Mini Plus** enables high-resolution imaging while eliminating physical disturbances such as cell damage or shaking. The automated system functions inside an incubator, eliminating the need to move samples in and out. Key system features: - Internal camera movement instead of a movable stage. - Steady vessel and cell sample positioning. - Stable growth environment. - Compatibility with various culture vessels. - High position reproducibility during multi-point imaging. **Celloger Mini Plus** provides reliable and stable imaging results for immunology research. ------------------------------------------------------------------------ ## References 1. Awasthi, Bhuwan Prasad, et al. (2021) Journal of Enzyme Inhibition and Medicinal Chemistry\ 2. Wu, Tsu-Tuan et al. (2009) Toxicology Letters\ 3. Saxena, Rajiv K et al. (2003) Journal of Biosciences\ 4. Taciak, Bartłomiej, et al. (2018) PLoS One\ 5. Ariganello, Marianne B., et al. (2018) International Journal of Nanomedicine\ 6. Manda-Handzlik, Aneta, et al. (2018) Immunology and Cell Biology\ 7. Riss, Terry, et al. (2019) Assay Guidance Manual \[Internet\]\ 8. Takishita, Yutaka, et al. (2019) Journal of Clinical Biochemistry and Nutrition\ 9. Pal, Kunal, et al. (2019) Materials Science and Engineering: C\ 10. Diwu, Zhenjun, et al. (1999) Chemistry & Biology ------------------------------------------------------------------------ **FOR RESEARCH USE ONLY** and not for use in diagnostic procedures.\ Copyright © 2022, by CURIOSIS Inc. All rights reserved.\ CRAN008-2022\ www.curiosis.com