Abstract
BackgroundMany cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists.ResultsWe report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples.ConclusionThe assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.
Highlights
Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed
We hypothesised that: 1) permeabilisation could be defined in terms of a general macromolecule size feature, such as molecular weight (MW). 2) An intrinsic cellular factor could serve as an internalisation marker for molecules of different MW
Since cell internalisation is directly proportional to molecule size [2], our results suggest that evaluation of permeabilisation can be achieved by establishing MW cut-offs, where MW internalisation markers indicate the permeabilisation efficiency expected for biomolecules of similar size
Summary
Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. We hypothesised that an accessible method enabling the assessment of cell permeabilisation, in terms of MW cut-offs and applicable to different cell types, could facilitate cell permeabilisation assessment Such a method could be enabled via the exploitation of intrinsic cellular motifs as targets for reporter molecules of a given size. SAv is a globular tetramer, with a MW of ~ 52 KDa, and a dimension of 5 nm, with each monomer able to interact with a biotin molecule [16] With this information, we developed a new assay to assess permeabilisation, using flow cytometry as a readout. The detection of naturally biotinylated intracellular proteins by SAv serves as a MW marker for cell internalisation This assay can be adapted for research on different biomolecules in eukaryotic or prokaryotic cells and is scalable to high-throughput settings
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