Detecting Insulin-Specific CD4+ T Cells

Insulin-specific CD4⁺ T cells were detected using insB_10-23r3:I-A^g7 tetramer reagent and dual color staining with magnetic enrichment as described ^{[24 (link), 25 (link)]}. Briefly, single cell suspensions were incubated for one hour at room temperature with 10 nM of tetramers conjugated to phycoerythrin (PE) and allophycocyanin (APC) in medium containing Fc receptor block (2.4G2) and 0.05% sodium azide. Spleen and non-draining lymph node samples were subjected to magnetic enrichment following a 30 minute incubation with both anti-PE and anti-APC microbeads at 4°C (Miltenyi Biotec). Single cell suspensions from the pancreas were isolated using collagenase P digestion (Roche) and discontinuous Percoll gradients (44%/67%) ^{[24 (link), 25 (link)]}. Following tetramer staining, single cell suspensions from pancreas and pancreatic lymph node were subjected to surface staining for flow cytometric analysis.