Smart Science: How to visualize movements and behaviors of cells in real-time

Green fluorescent protein (GFP) and red fluorescent protein (RFP) are two of the most commonly used fluorescent proteins in scientific research. GFP and RFP are used to label cells, making them easier to detect and study. GFP and RFP are both proteins that emit fluorescence if exposed to specific wavelengths of light. By inserting these proteins into cells, researchers track #cellmigration, monitor #protein localization, and study #cellinteractions in real-time.

GFP is a protein derived from the jellyfish Aequorea victoria, and it emits a bright green light when exposed to certain wavelengths of light. GFP has been widely used in #cellbiology due to its bright green fluorescence and stability after insertion into cells. RFP is a protein derived from a different species of jellyfish, Discosoma sp., and it emits bright red light when exposed to certain wavelengths of light. RFP is exactly like GFP however it was developed by site directed mutagenesis of GFP, such approaches are used to improve the fluorescent proteins' brightness.

To label cells with GFP or RFP, a DNA construct containing the gene for the protein is first created. The construct is then inserted into cells using methods such as viral transduction, lipofection, electroporation, or microinjection. The labeled cells are then imaged under a fluorescence microscope, you cannot use a normal microscope as you need a dichroic mirror specific sets of excitation and emission filters to do fluorescent imaging.

Fluorescent proteins are assessed on the basis of a fluorophore's intrinsic fluorescence brightness which is proportional to the product of the molecular extinction coefficient(ε) and fluorescence quantum yield(Φ). Traditionally these proteins are excited by visible light however recently few proteins have been developed which are excited by infrared; this is beneficial as this allows greater penetration and minimal scattering of light while imaging⁶.

GFP-labeled cells to detect and analyze movements of cells

GFP-labeled cells can be used to detect and analyze the movements of cells, cellular organelles, and proteins in a laboratory setting. They provide a non-invasive method to directly visualize and track the movement of cells, providing a powerful tool for studying cell motility, migration, and interactions. Additionally, GFP-labeled cells can be used to track the development of specific cells or tissues, providing insight into the mechanisms of development and differentiation.

Furthermore, GFP-labeled cells can be used to analyze the effects of different drugs or treatments on a cell population. By monitoring the fluorescence of GFP-labeled cells, researchers can observe changes in cell behavior and protein expression in response to different treatments, providing insight into the mechanisms of drug action and toxicity.

In addition to GFP-labeled cells, RFP-labeled cells are also widely used in scientific research. These proteins can be used in conjunction with GFP-labeled proteins to study co-localization of proteins within cells. By labeling different proteins with different colors of fluorescent proteins, researchers can observe their co-localization in the same cell, providing insight into protein interactions and complex cellular processes.

GFP-labeled cells provide a powerful tool for studying cell behavior, development, and drug responses, while RFP-labeled cells can be used to study protein interactions and localization within cells. These fluorescent labeling techniques have revolutionized the field of cell biology, providing non-invasive methods to directly visualize and track the behavior of cells and proteins, and leading to important insights into cellular mechanisms and behavior.

A researcher can use fluorescent proteins for isolation of specific cell types with differing gene expression patterns by FACS ^1,2 , brainbow to visualize the connectome ³, detect changes in the cell health and metabolism ^4,5.

GFP-labeled cells to analyze cell interactions in a coculture

In cell biology, a coculture system refers to the in vitro cultivation of two or more cell types in the same environment. By using labeled cells, researchers can track and quantify the interactions between these cells, providing a powerful tool for studying complex cellular processes and behaviors.

Labeled cells are cells that have been marked with a unique identifier, such as a fluorescent dye or a radioactive isotope. By labeling cells with different markers, researchers can distinguish one cell type from another and track their interactions over time.

In a coculture system, labeled cells can be introduced to the environment and allowed to interact with one another. As the cells interact and communicate, researchers can monitor and quantify the interactions between them. This can provide insight into cellular behaviors and mechanisms that would be difficult to observe in other experimental systems.

For example, in a coculture system that includes immune cells and cancer cells, labeled immune cells can be tracked as they interact with the cancer cells. Researchers can observe how the immune cells recognize and respond to the cancer cells. This information can help researchers better understand the mechanisms underlying the immune response to cancer, and may ultimately lead to the development of new immunotherapies. Cocultre has been used for other cell types such as HUVECs, OB-like cells, osteoblasts, endothelial cells, mouse embryonic stem cells, embryonic neuronal tissue, granule cells, pyramidal cells, interneurons and Jurkat cells^{7, 8, 9, 10}.

Monitoring embryonic stem cells

Not only are GFP and RFP labeling safe and non-toxic, but they have also revolutionized cell biology research by allowing scientists to study cells in new and innovative ways. These proteins are essential tools for understanding cellular behavior and gaining insights into how cells work and interact with each other. GFP-labeled cells have been extensively used to study embryonic stem cells (ESCs). GFP-labeled ESCs provide a non-invasive method to study the pluripotent potential of these cells, as well as to track their behavior during differentiation and in vivo transplantation.

GFP-labeled ESCs can be used to track the expression of pluripotency markers, such as Oct4, Sox2, and Nanog, which are crucial for maintaining the undifferentiated state of ESCs. GFP-labeled ESCs can be visualized using fluorescence microscopy, allowing for the direct observation of their undifferentiated state and differentiation potential. By monitoring GFP-labeled ESCs, researchers can determine the effects of different culture conditions or genetic modifications on ESC behavior and differentiation potential.

Furthermore, GFP-labeled ESCs can be used to study lineage commitment and differentiation. By introducing GFP-labeled genes for lineage-specific markers, such as GFP-labeled beta-galactosidase for neural markers or GFP-labeled alpha-fetoprotein for hepatic markers, researchers can track the differentiation of ESCs towards specific lineages. GFP-labeled ESCs can also be used to generate chimeric animals, allowing for the tracking of ESC behavior and contribution to various tissues during development.

Finally, GFP-labeled ESCs can be used to study the behavior of ESCs after transplantation into host organisms. GFP-labeled ESCs can be visualized in vivo using fluorescence microscopy, allowing for the tracking of their behavior and integration into the host environment. GFP-labeled ESCs can also be used to study the potential risks of ESC-based therapies, such as tumorigenesis, by monitoring the behavior of GFP-labeled ESCs after transplantation. GFP labeled ESCs have been used to study primordial germ cell generation, chimeric fetuses and mammary duct morphogenesis ^{11, 12, 13 ,14, 15}.

So green fluorescent protein (GFP) and red fluorescent protein (RFP) labeling could be the solution for your research. Have a look at our catalog and call us to find out what can work for your project.

->Go to Catalog of our tagged Cells

#cellbiology #GFP #RFP #fluorescence #cellularresearch #research

References

1. Smith, M., Culhane, A., Donovan, M. et al. Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification. Br J Cancer 100, 1452–1464 (2009). https://doi.org/10.1038/sj.bjc.6604931
2. Crouch, E., Doetsch, F. FACS isolation of endothelial cells and pericytes from mouse brain microregions. Nat Protoc 13, 738–751 (2018). https://doi.org/10.1038/nprot.2017.158
3. Lichtman, J., Livet, J. & Sanes, J. A technicolour approach to the connectome. Nat Rev Neurosci 9, 417–422 (2008). https://doi.org/10.1038/nrn2391
4. Zhang, J., Wang, X., Cui, W. et al. Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage. Nat Commun 4, 2157 (2013). https://doi.org/10.1038/ncomms3157
5. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98. doi: 10.1016/j.cell.2007.12.033. PMID: 18267078.
6. Matlashov, M.E., Shcherbakova, D.M., Alvelid, J. et al. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nat Commun 11, 239 (2020). https://doi.org/10.1038/s41467-019-13897-6
7. Wang J, Liu X, Qiu Y, Shi Y, Cai J, Wang B, Wei X, Ke Q, Sui X, Wang Y, Huang Y, Li H, Wang T, Lin R, Liu Q, Xiang AP. Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells. J Hematol Oncol. 2018 Jan 22;11(1):11. doi: 10.1186/s13045-018-0554-z. PMID: 29357914; PMCID: PMC5778754.
8. Osorio B, León U, Galván EJ, Gutiérrez R. Cocultures of GFP(+) -granule cells with GFP(-) -pyramidal cells and interneurons for the study of mossy fiber neurotransmission with paired recordings. Hippocampus. 2013 Apr;23(4):247-52. doi: 10.1002/hipo.22102. Epub 2013 Feb 25. PMID: 23436451.
9. Glavaski-Joksimovic A, Thonabulsombat C, Wendt M, Eriksson M, Palmgren B, Jonsson A, Olivius P. Survival, migration, and differentiation of Sox1-GFP embryonic stem cells in coculture with an auditory brainstem slice preparation. Cloning Stem Cells. 2008 Mar;10(1):75-88. doi: 10.1089/clo.2007.0065. PMID: 18241123.
10. Shahabipour F, Oskuee RK, Dehghani H, Shokrgozar MA, Aninwene GE 2nd, Bonakdar S. Cell-cell interaction in a coculture system consisting of CRISPR/Cas9 mediated GFP knock-in HUVECs and MG-63 cells in alginate-GelMA based nanocomposites hydrogel as a 3D scaffold. J Biomed Mater Res A. 2020 Aug 1;108(8):1596-1606. doi: 10.1002/jbm.a.36928. Epub 2020 Mar 27. PMID: 32180319.
11. Wen B, Li R, Cheng K, Li E, Zhang S, Xiang J, Wang Y, Han J. Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues. Sci Rep. 2017 Jun 8;7(1):3030. doi: 10.1038/s41598-017-02783-0. PMID: 28596585; PMCID: PMC5465063.
12. Song J, Ding F, Li S, Peng S, Zhu Y, Xue K. Prolactin stimulation affects the stem cell-dependent mammary repopulating ability of embryonic mammary anlagen. Int J Dev Biol. 2018;62(9-10):623-629. doi: 10.1387/ijdb.180109kx. PMID: 30378386.
13. Ma X, Li P, Sun X, Sun Y, Hu R, Yuan P. Differentiation of female Oct4-GFP embryonic stem cells into germ lineage cells. Cell Biol Int. 2018 Apr;42(4):488-494. doi: 10.1002/cbin.10918. Epub 2018 Jan 17. PMID: 29271529.
14. Perez-Cunningham J, Boyer SW, Landon M, Forsberg EC. Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene. Exp Hematol. 2016 Aug;44(8):755-764.e1. doi: 10.1016/j.exphem.2016.05.002. Epub 2016 May 13. PMID: 27185381; PMCID: PMC4962998.