title: Development of a bioluminescent ATP assay to quantify mammalian and bacterial cell number from a mixed population.
abstract: Modification of biomaterial surfaces is one approach aimed at improving cellular interactions and the subsequent integration of medical devices into the body. However, by optimising surfaces to enhance mammalian cell adhesion there is the potential risk that adhesion of bacteria will also be increased. Bacterial colonisation of biomaterials can be problematic as infection often results in morbidity and the consequent removal of the failing device from the body. Currently, quantifying cellular adhesion from a mixed population of bacterial and mammalian cells can only be determined by slow and laborious methods such as microscopy. ATP is a key molecule in the metabolism of both mammalian and bacterial cells and can be used to quantify cell numbers. In this study, we have modified a bioluminescence-based ATP assay to enable the differential measurement of both mammalian and bacterial cell ATP levels within the same culture. Mixed populations of Staphylococcus epidermidis and 3T3 fibroblasts were assessed both in suspension and adhered to a surface. ATP levels from cultures in suspension were selectively extracted and measured from both cell types, revealing a linear trend that would enable the differentiation of cell numbers from a mixed population. The application of the assay to adhered mixed cultures also allowed differences in ATP levels from both cell types to be distinguished. The data presented reveals that this assay would be useful for the rapid screening of cellular adhesion to modified surfaces although, its use in detecting subtle differences in ATP levels may be limited due to natural interactions between the two cell types.
authors.affiliation: School of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Dexter
authors.forename: Miguel
authors.initials: M
authors.lastname: Cámara
authors.forename: Martyn
authors.initials: M
authors.lastname: Davies
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Shakesheff
mesh_terms: D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000032:analysis*; D000818:Animals; D001422:Bacterial Adhesion; D001672:Biocompatible Materials; D002448:Cell Adhesion; D002452:Cell Count / Q000379:methods*; D015169:Colony Count, Microbial / Q000379:methods*; D008163:Luminescent Measurements; D008422:Materials Testing; D051379:Mice; D018613:Microscopy, Confocal; D013212:Staphylococcus epidermidis / Q000166:cytology / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D000255:Adenosine Triphosphate