GAD65-Specific CD8+ T Cell Quantification

On day 6, following previously published protocols [37 (link),38 ], approximately 5x10⁵ cells, stimulated with the GAD65 AA 114–122 peptide (supplemented with IL-2) or cultured with IL-2 alone, were washed and finally resuspended in approximately 50 μl. 1 μl of phycoerithrin (PE) labelled GAD65 AA 114–122 pentamer was added to each cell preparation and incubated in ice for 30 minutes in the dark, then washed in wash buffer. Mouse monoclonal antibody (mAb) anti-human CD8 at 1:20 dilution (fluorescein (FITC) labelled, clone HIT8a, cat# 555634, Becton & Dickinson (BD), Pharmingen, San Diego, CA, USA) and mouse mAb anti-human CD3 at 1:20 dilution (allophycocyanin (APC) labelled, clone UCHT1, cat# 555335, BD, Pharmingen) were added for further discriminating the different cell preparations. After staining, cells were immediately acquired for the analysis on the FACSCanto II (BD). Flow cytometric profiles were analyzed using the FACSDiva software (BD Bioscience). Dead cells were excluded from the analysis by side/forward scatter gating [39 (link)]. A minimum of twenty thousand events, gated on living cells, were collected per dataset.