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Method for determining of cytotoxicity based on the release of fluorescent proteins

  • 0Institute of Future Biophysics, Institutskiy per. 9, Dolgoprudny, Moscow Oblast, Moscow, Russia. dmtlifnv@gmail.com.
Bmc Molecular and Cell Biology +

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January 28, 2025

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January 28, 2025

View abstract on PubMed

Summary

This summary is machine-generated.

This study presents a novel cytotoxicity assay using fluorescent proteins (GFP and RFP) to detect dead cells, offering an alternative to the LDH test. The method accurately assesses compound toxicity in various 3D culture models.

Area Of Science

  • Cell Biology
  • Toxicology
  • Biotechnology

Background

  • Cytotoxicity assays are crucial for drug development and toxicology.
  • Existing methods like the lactate dehydrogenase (LDH) test may require chromogenic substrates.
  • Assessing cell viability in complex 3D cultures and cocultures presents challenges.

Purpose Of The Study

  • To develop and validate a novel cytotoxicity assay utilizing fluorescent proteins (GFP and RFP).
  • To compare the performance of this assay in different cell culture formats, including 2D monolayers, spheroids, and 3D alginate hydrogels.
  • To evaluate the assay's utility in coculture models and assess differential cytotoxicity.

Main Methods

  • Detection of released green fluorescent protein (GFP) and red fluorescent protein (RFP) from dead cells in culture medium.
  • Application of the assay to classical monolayer cultures, spheroids, and 3D alginate hydrogel cultures.
  • Utilization of capecitabine as a model cytotoxic agent in cocultures with liver cells (Huh7) and NCI-H1299 cells.

Main Results

  • The fluorescent protein release assay provides a direct measure of cell death without requiring a chromogenic substrate.
  • The method successfully quantified cytotoxicity in various 3D culture models.
  • Coculture experiments demonstrated the ability to independently assess the viability of different cell lines and revealed differential sensitivity to capecitabine based on 3D culture type.

Conclusions

  • The developed fluorescent protein-based cytotoxicity assay is a sensitive and versatile tool for evaluating chemical compound toxicity.
  • This method offers advantages over traditional assays, particularly in complex 3D and coculture systems.
  • The findings highlight the importance of culture format in determining drug sensitivity and underscore the assay's potential for drug discovery and toxicological studies.

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