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Related Experiment Video

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Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
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A novel cell permeability assay for macromolecules.

Yensi Flores Bueso1,2,3, Sidney Walker1,2,3, Jennifer Quinn1

  • 1CancerResearch@UCC, University College Cork, Cork, Ireland.

BMC Molecular and Cell Biology
|October 31, 2020
PubMed
Summary

Researchers developed a novel assay using Streptavidin (SAv) to measure cell permeability for molecules of different sizes in both mammalian and bacterial cells. This method aids in developing host DNA depletion strategies for microbiome analysis.

Keywords:
Biotin-streptavidinCell permeabilisationHost DNA depletionIn vitro labellingMW marker

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Area of Science:

  • Cell biology
  • Microbiology
  • Molecular biology

Background:

  • Established cell permeabilization methods lack size-specificity for both mammalian and bacterial cells.
  • A need exists for a versatile assay to assess macromolecule internalisation across different cell types.

Purpose of the Study:

  • To develop a size-specific cell permeability assay applicable to both eukaryotic and prokaryotic cells.
  • To utilize Streptavidin (SAv) as a molecular weight marker for assessing internalisation.
  • To evaluate this assay for host DNA depletion strategies in microbiome analysis.

Main Methods:

  • Developed an assay using intrinsically biotinylated cell components and Streptavidin (SAv) as a reporter.
  • Employed flow cytometry for quantitative readout of SAv-conjugate internalisation.
  • Tested the assay on formalin-fixed samples for host DNA depletion strategy development.

Main Results:

  • Formalin fixation does not create pores permeable to 60 KDa molecules in mammalian or bacterial cells.
  • Saponin demonstrated selective mammalian cell permeabilisation among tested surfactants.
  • Optimized host DNA depletion using Saponin and Benzonase nuclease for formalin-fixed samples.

Conclusions:

  • The developed Streptavidin-based assay offers a sensitive and accessible tool for evaluating cell membrane permeability to macromolecules.
  • This assay is valuable for size-specific permeability assessments in diverse cell types.
  • The findings contribute to improved host DNA depletion methods for microbiome research.