IgM binds strongly to permeabilised non-apoptotic and apoptotic thymocytes.

<p>Non-apoptotic and apoptotic mouse thymocytes, induced by overnight culture, were permeabilised with saponin prior to incubation with Balb/c plasma. Permeability was confirmed by PI staining and IgM binding was assessed by flow cytometry. For confocal microscopy AnnV staining was used to indicate permeabilisation <b>A</b>) Following permeablisation non-apoptotic thymocytes were predominantly PI positive. Non-permeable non-apoptotic thymocytes remained consistently IgM negative, whilst the majority of permeable thymocytes were IgM positive. Inclusion of isotype control IgM antibodies (mouse (Balb/c) IgM κ isotype control) confirmed that IgM binding was not due to a non-specific interaction of IgM. <b>B</b>) A small proportion of apoptotic thymocytes were PI positive representing late apoptotic thymocytes; however following permeabilisation with saponin the cell population became PI positive. Whilst a minority of apoptotic thymocytes were IgM positive, representing the AnnV^<b>+</b>IgM^<b>+</b> population discussed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131849#pone.0131849.g001" target="_blank">Fig 1C–1E</a>, with permeabilisation the population became IgM positive. <b>C</b>) Confocal microscopy demonstrates that permeabilised thymocytes were AnnV positive, used to indicate permeability, with IgM appearing to bind part of the cellular membrane. Representative histograms (Non-apoptotic thymocytes <i>n</i> = 3; Apoptotic thymocytes <i>n</i> = 4). Images were taken using a Leica SP5 with an x63/1.4 objective. Whole image contrast/brightness was adjusted using ImageJ or Adobe Photoshop.