Background Many 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 all cells exists. Results We report for the first time, 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

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... marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here in the development of host DNA depletion strategies for microbiome analysis of formalin-fixed samples. This strategy requires 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 FF does not generate pores which allow the introduction of 60 KDa molecules in both mammalian and bacterial membranes/envelopes. Among surfactants tested, Saponin 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 host depletion strategy for formalin fixed samples. Conclusion The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.