Overcoming Protein A-driven Nonspecific Antibody Staining of S. aureus in Immunofluorescence Microscopy

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Abstract
The ability to detect host cell factors during Staphylococcus aureus infection in vitro by immunofluorescence microscopy is severely hampered by staphylococcal protein A (SpA), a cell wall-anchored protein that binds the fragment crystallizable (Fc) region of immunoglobulins. This interaction generates strong nonspecific fluorescent signals on the bacterial surface, complicating data interpretation and limiting the accuracy of quantitative image analysis. Several measures have been put forward to overcome this obstacle, most importantly the pre-incubation with an anti-SpA antibody (αSpA) and the use of human serum (HS) as blocking agent and antibody diluent. To highlight this feature to general fluorescence microscopy users, we here systematically evaluated these two strategies. Using S. aureus coated on coverslips and S. aureus -infected A549 cells, we highlight the efficiencies of both approaches to markedly reduce nonspecific fluorescence, with HS treatment yielding the most profound suppression. Notably, HS, containing high levels of human immunoglobulins, offered a robust, cost-effective and broadly applicable solution for minimizing SpA-driven artifacts, thereby improving immunofluorescence microscopy in S. aureus infection models in vitro .
Funding Information
  • Deutsche Forschungsgemeinschaft (Germany´s Excellence Strategy – EXC 2051 – Project-ID 390713860)
  • Deutsche Forschungsgemeinschaft (SFB 1278/2 PolyTarget-316213987-A04, -D01,-D02, -Z01)
  • Deutsche Forschungsgemeinschaft (GRK M-M-M: GRK 2723/1 – 2023 – ID 44711651)
  • Deutsche Forschungsgemeinschaft (GRK PhInt: GRK 3014/1)
  • Deutsche Forschungsgemeinschaft (Instrument funding modular STED INST 1757/25-1 FUGG)
  • Deutsche Forschungsgemeinschaft (Instrument funding ID 460889961 multi-photon laser scanning device)
  • Thüringer Minesterium für Bildung, Wissenschaft und Kultur
  • Leibniz Association (Leibniz ScienceCampus InfectoOptics Jena, project number W8/2018)
  • Leibniz Association (Leibniz Collaborative Excellence Programme, project AMPel, project number K548/2023)
  • Freistaat Thüringen (AdvancedSTED / FGZ: 2018 FGI 0022)
  • Freistaat Thüringen (Advanced Flu-Spec / 2020 FGZ: FGI 0031)
  • Freistaat Thüringen (Multi-XUV / 2023 FGR 0054)
  • Freistaat Thüringen (2018 IZN 0002 (Thimedop))
  • Bundesministerium für Wirtschaft und Energie (ZIM, project 16KN070934 / Lab-on-a-chip FCS-Easy)
  • Bundesministerium für Wirtschaft und Energie (ZIM, project 16KN070967 / Lab-on-a-chip SMARTIES)
  • Photonics Research Germany (FKZ: 13N15713 / 13N15717)
  • European Union (European Regional Development Fund (EFRE))

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