Handpicked islets were pooled in 24-well plates for 24 h in RPMI supplemented with 10% FCS, penicillin–streptomycin, and 5.10−5 M β-mercaptoethanol allowing extrusion of infiltrating lymphocytes (25 (link)). Infiltrating lymphocytes were recovered. Cells recovered were used for the analysis of T cells and antigen-presenting cells. T-cell analysis was performed using the Foxp3 intra-staining buffer set according to the recommendation of the manufacturer (eBioscience) with the following combination: anti-mouse CD45-efluo450, anti-mouse CD3ϵ-AlexaFluo700, anti-mouse B220-HorizonViolet500, anti-mouse CD8α-Percp-Cy5.5, anti-mouse CD4-PE, anti-mouse Foxp3-APC, and anti-mouse CD25-BrillantViolet711 mAbs (eBioscience). β Cells and dendritic cells were analyzed with anti-mouse CD45-AlexaFluo700, anti-mouse TCRβ-APC-Cy7, anti-mouse B220-HorizonViolet500, anti-mouse CD11b-efluor450, anti-mouse CD11c-APC, anti-mouse CD8α-BrillantViolet605, and anti-mouse CD4-PE mAbs (eBioscience). Acquisitions were performed with a BD LSR-Fortessa flow cytometer and analyzed using FlowJo 10.7.1 software.
Luce S., Guinoiseau S., Gadault A., Letourneur F., Nitschke P., Bras M., Vidaud M., Charneau P., Larger E., Colli M.L., Eizirik D.L., Lemonnier F, & Boitard C. (2021). A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes. Frontiers in Immunology, 12, 748679.
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