System:
Celloger
The Live Cell Imaging is a method to examine living cells over a period of time using images acquired by time-lapse microscopy. Real time imaging of cellular processes such as cell migration, development and trafficking can be employed as an important tool for research in various academic fields including cell biology, cancer research, neuroscience, pharmacology and developmental biology. In order to observe the cells in a live state, incubator function is added to cover the microscope to control carbon dioxide, temperature and humidity. But in many cases, controlling the temperature and humidity suitable for cell growth is challenging. To overcome such shortcomings, affordable and compact imaging devices that can be put into a cell culture incubator are being developed. Using the Live Cell Imaging is possible to visualize the dynamic cellular events using Bright-field or Fluorescence Microscopy and to study cellular behavior such as cell division, migration, signaling, and interactions with other cells or molecules.
The Celloger Series
An innovative Live Cell Imaging line developed by Curiosis. With its exceptional image quality and unmatched convenience, it empowers researchers with advanced features. The Celloger equipment have compact size and it can be placed inside a standard cell culture incubator (Figure 1). By simply placing the device within the incubator and connecting it to an external PC, researchers are able to remotely observe cells in real-time. The time-lapse function permits the capture of cell images according to the schedule set up and can be easily converted into time-lapse video. Using the Celloger series is possible to obtain clear bright-field images using contrast-enhanced optics and fluorescence images of live cells in real time with a minimum light intensity by optimizing fluorescence filter and light path.
Figure 1: Celloger Mini-Plus (2 units) inside CO2 incubator.
Cytotoxicity assay
Several staining reagents that measure the degree of cell death using a phenomenon in which the integrity of the cell membrane is damaged and the cell permeability is increased during the cell death are commercially available. To measure the cytotoxicity by nocodazole, dead cells were stained with green fluorescent CellToxâ„¢ dye. Using the Celloger it was confirmed that the number of cells measured by fluorescence increased as well the cell permeability due to cell death after 20 hours (Figure 2).
Figure 2: (A) Cell image after 35 hours from the treatment with
62.5nM nocodazole (Scale bar, 200um). (B) Fluorescence coverage by hour.
Apoptosis assay
Apoptosis is the process of programmed cell death where processes such as membrane blebbing, cell shrinkage and nuclear fragmentation occur.
It was found that fluorescent materials were released and detected after cleavage of the peptide DEVD caused by the treatment with Staurosporine, a material known to activate caspase and cause apoptosis (Figure 3).
Figure 3: Using fluorescence detection of activated caspase to quantify apoptosis of Hela cells (Scale bar, 200μm). The images were collected every 30 min using the Celloger Nano for 15 hours and 30 minutes.
Transfection assay
Transfection is a modern and powerful method used to insert foreign nucleic acids into eukaryotic cells. Real-time cell imaging is considered to be used well in many applications for quantifying cell transfection efficiency or monitoring the effect of transfected genes.
The fluorescence with the expression of green fluorescence protein in pCMV-GFP vector transfected in a cell was observed every 2 hours through Celloger Nano and it was confirmed that the green fluorescence protein started to be expressed from 4 hours after transfection and it was maintained until 16 hours after transfection (Figure 4).
Figure 4: Time-lapse image of EGFP expression following pCMV GFP
plasmid transfection (Scale bar, 200μm). The images were collected every 2 hr for 40 hr.
Cell monitoring
The changes in cell morphology occur at every major point in cell cycle and monitoring these changes in the appearance of cells in real-time is very important. By monitoring cell morphology (Figure 5) in real time researchers can detect the signs of contamination in earlier stage, senescence stage of the cells, and define the best time for subculture or harvest.
Figure 4: Time-lapse image of EGFP expression following pCMV GFP
plasmid transfection (Scale bar, 200μm). The images were collected every 2 hr for 40 hr.
Wound healing assay
Wound healing assay is the easiest and fastest way to check cell migration. When a scratch or space is created in the monolayer of cells, they show the process of movement to fill in the wound until the wound is entirely healed with the new healthy cells. Using time-lapse imaging of Celloger it is possible to analyze the wound healing events easily and effectively (Figure 6).
Figure 6: Wound healing image of HeLa cells.
Cell Proliferation
Cell proliferation is to quantify the increasing number of cells over a period of time to verify that the cells are growing in normal growth process.
As a method of quantification, number of fluorescent dyed cells or cell confluency is measured. In other words, a graph of cell number or confluency changes over time is mainly used as the result of proliferation.
Figure 7: Cell proliferation (NIH/3T3 cell).
Real-time monitoring of spheroid cytotoxicity
2D cultures are widely used because of their cost-efficiency and convenience. Nevertheless, the limitations of 2D culture systems, such as the loss of cell-to-cell or cell-to-matrix interactions and tissue-specific structures, hinder their capacity to mimic in vivo conditions, especially in disease models like cancer. Due to these limitations, there is a growing interest in 3D culture systems that provide more realistic model resembling a complex in vivo environment.
The study presented herein highlights the power of Celloger Pro, a cutting-edge live cell imaging system, in investigating the effects of an anticancer drug, Staurosporine (SSP), on 3D spheroids made from HEK293-GFP stable cells. This application note demonstrating its capacity to dynamically capture and quantify cellular responses to drug treatment in a three-dimensional context.
Figure 8: The results of spheroid cells with 0, 0.1, and 1 μM of SSP. (A) Merged of green and red fluorescence images for each concentration of SSP (scale bar: 200 μm). (B) Brightfield images with spheroid’s diameter (scale bar: 200 μm). (C) Comparative graph of spheroid area at 0 and 24 hours for each concentration of SSP. n=3 for each group. ***P < 0.0001 (D) Relative red fluorescence intensity graph over time.
References
1. Jordan, M. A., Thrower, D., & Wilson, L. (1992). Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. Journal of cell science, 102 (1), 401-416.
2. Blajeski, A. L., Phan, V. A., Kottke, T. J., & Kaufmann, S. H. (2002). G 1 and G 2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells. The Journal of clinical investigation, 110 (1), 91-99.
3. Jordan, M. A., Thrower, D., & Wilson, L. (1992). Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. Journal of cell science, 102 (1), 401-416.
4. Blajeski, A. L., Phan, V. A., Kottke, T. J., & Kaufmann, S. H. (2002). G 1 and G 2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells. The Journal of clinical investigation, 110 (1),91-99.
5. Fang, Ye, and Richard M. Eglen. “Three-dimensional cell cultures in drug discovery and development.” Slas discovery: Advancing Life Sciences R&D 22.5 (2017): 456-472.
6. Ravi, Maddaly, et al. “3D cell culture systems: advantages and applications.” Journal of cellular physiology 230.1 (2015): 16-26.
