Multiparameter Flow Cytometry Analysis

Seven independent experiments with a total of 20 animals (wt n=11, INS^C94Y tg n=9) were performed. Staining of 2 x 10⁵ cells per well was performed with mouse anti-human CD79a (clone HM57, Bio-Rad AbD Serotec, Puchheim, Germany, 1:100) for identification of B cells and Alexa Fluor 647-conjugated rat anti-human CD3ϵ [clone CD3-12, 1:200; cross-reactive to pig (16 (link))] for identification of T cells. We used FITC-conjugated mouse anti-pig CD4α (clone MIL17, Bio-Rad AbD Serotec, Puchheim, Germany, 1:20) and Alexa Fluor 647-conjugated mouse anti-pig CD8α (clone 76-2-11, Becton Dickinson, Heidelberg, Germany, 1:400) to identify αβ T cells and mouse anti-pig SWC5 (clone b37c10, Bio-Rad AbD Serotec, Puchheim, Germany, 1:100) for identification of γδ T cell subpopulation. If necessary, a secondary antibody was used (Alexa Fluor 647-conjugated goat F(ab’)2 anti-mouse IgG (Fc), Dianova, Hamburg, Germany, 1:1000). Dead cells were excluded via labeling with Viobility 405/520 Fixable Dye (Miltenyi Biotec, Bergisch Gladbach, Germany) prior to the various stainings mentioned above. Analyses were performed with MACSQuant Analyzer 10 and Flowlogic Software (both Miltenyi Biotec, Bergisch Gladbach, Germany).