In vivo mouse micronucleus acridine orange staining and HCR generation: Protocol optimisation

UKEMS 2025 -- The OECD 474 guideline states that the use of a DNA specific stain [e.g., acridine orange (A/O) (35) or Hoechst 33258 plus pyronin-Y)] can eliminate some of the artifacts associated with using a non-DNA specific stain. At Labcorp Harrogate, the in vivo mouse bone marrow micronucleus (MN) assay has traditionally been run using Giemsa to stain bone marrow preparations rather than the higher affinity DNA binding A/O. Historically, when looking at A/O as a stain for mouse bone marrow preparations, excessive fluorescence with nucleated cells can create difficulty for the accurate analysis of the immature cell populations. In addition, the use of cellulose filtration (to remove the majority of nucleated cells) has left little bone marrow cellular material remaining to allow for any potential back-up analysis, or for follow-up mechanistic assessment [fluorescence in situ hybridization (FISH) or immunofluorescent antikinetochore staining (CREST)]. However, following method optimisation, we have been able to generate strong historical control data (vehicle and positive control) for male and female mice using A/O staining. C-charts and stability index calculations illustrate good control of the data through five different experimental occasions confirming suitability of the optimised protocol. In addition, we have adjusted our cellulose filtration method to allow a greater recovery of cellular material, allowing suitable slide preparation for initial analysis, any potential back-up analysis and slides for CREST/FISH mechanistic work.