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Fluorescence intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of cell differentiation in the small intestinal organoids using cholera toxin

Irina Okkelman (UGent) and Ruslan Dmitriev (UGent)
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Abstract
Live cell microscopies of in vitro, ex vivo, and in vivo experimental intestinal models enable visualizing cell proliferation, differentiation, and functional cellular status in response to intrinsic and extrinsic (e.g., in the presence of microbiota) factors. While the use of transgenic animal models expressing biosensor fluorescent proteins can be laborious and not compatible with clinical samples and patient-derived organoids, the use of fluorescent dye tracers is an attractive alternative. In this protocol, we describe how the differentiation-dependent intestinal cell membrane composition can be labeled using fluorescent cholera toxin subunit B (CTX) derivatives. By using the culture of mouse adult stem cell-derived small intestinal organoids, we show that CTX can bind specific plasma membrane domains in differentiation-dependent manner. Green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescent CTX derivatives also display additional contrast in a fluorescence lifetime domain, when probed by the fluorescence lifetime imaging microscopy (FLIM), and can be used together with other fluorescent dyes and cell tracers. Importantly, CTX staining remains confined to specific regions in the organoids after fixation, which enables using it in both live cell and fixed tissue immunofluorescence microscopies.
Keywords
Cholera toxin, Intestinal epithelium, Intestinal organoid, FLIM, Live cell imaging

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MLA
Okkelman, Irina, and Ruslan Dmitriev. “Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.” Intestinal Differentiated Cells : Methods and Protocols, edited by Paloma Ordonez-Moran, vol. 2650, Humana, 2023, pp. 171–95, doi:10.1007/978-1-0716-3076-1_14.
APA
Okkelman, I., & Dmitriev, R. (2023). Fluorescence intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of cell differentiation in the small intestinal organoids using cholera toxin. In P. Ordonez-Moran (Ed.), Intestinal differentiated cells : methods and protocols (Vol. 2650, pp. 171–195). https://doi.org/10.1007/978-1-0716-3076-1_14
Chicago author-date
Okkelman, Irina, and Ruslan Dmitriev. 2023. “Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.” In Intestinal Differentiated Cells : Methods and Protocols, edited by Paloma Ordonez-Moran, 2650:171–95. New York, NY: Humana. https://doi.org/10.1007/978-1-0716-3076-1_14.
Chicago author-date (all authors)
Okkelman, Irina, and Ruslan Dmitriev. 2023. “Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.” In Intestinal Differentiated Cells : Methods and Protocols, ed by. Paloma Ordonez-Moran, 2650:171–195. New York, NY: Humana. doi:10.1007/978-1-0716-3076-1_14.
Vancouver
1.
Okkelman I, Dmitriev R. Fluorescence intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of cell differentiation in the small intestinal organoids using cholera toxin. In: Ordonez-Moran P, editor. Intestinal differentiated cells : methods and protocols. New York, NY: Humana; 2023. p. 171–95.
IEEE
[1]
I. Okkelman and R. Dmitriev, “Fluorescence intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of cell differentiation in the small intestinal organoids using cholera toxin,” in Intestinal differentiated cells : methods and protocols, vol. 2650, P. Ordonez-Moran, Ed. New York, NY: Humana, 2023, pp. 171–195.
@incollection{01H45HDJV1HBXH0ZCVBE09QR1K,
  abstract     = {{Live cell microscopies of in vitro, ex vivo, and in vivo experimental intestinal models enable visualizing cell proliferation, differentiation, and functional cellular status in response to intrinsic and extrinsic (e.g., in the presence of microbiota) factors. While the use of transgenic animal models expressing biosensor fluorescent proteins can be laborious and not compatible with clinical samples and patient-derived organoids, the use of fluorescent dye tracers is an attractive alternative. In this protocol, we describe how the differentiation-dependent intestinal cell membrane composition can be labeled using fluorescent cholera toxin subunit B (CTX) derivatives. By using the culture of mouse adult stem cell-derived small intestinal organoids, we show that CTX can bind specific plasma membrane domains in differentiation-dependent manner. Green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescent CTX derivatives also display additional contrast in a fluorescence lifetime domain, when probed by the fluorescence lifetime imaging microscopy (FLIM), and can be used together with other fluorescent dyes and cell tracers. Importantly, CTX staining remains confined to specific regions in the organoids after fixation, which enables using it in both live cell and fixed tissue immunofluorescence microscopies.}},
  author       = {{Okkelman, Irina and Dmitriev, Ruslan}},
  booktitle    = {{Intestinal differentiated cells : methods and protocols}},
  editor       = {{Ordonez-Moran, Paloma}},
  isbn         = {{9781071630754}},
  issn         = {{1064-3745}},
  keywords     = {{Cholera toxin,Intestinal epithelium,Intestinal organoid,FLIM,Live cell imaging}},
  language     = {{eng}},
  pages        = {{171--195}},
  publisher    = {{Humana}},
  series       = {{Methods in Molecular Biology}},
  title        = {{Fluorescence intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of cell differentiation in the small intestinal organoids using cholera toxin}},
  url          = {{http://doi.org/10.1007/978-1-0716-3076-1_14}},
  volume       = {{2650}},
  year         = {{2023}},
}

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