Cellular immunoblotting has been validated multiple times to be able to distinguish type 1 diabetes patients from controls in blinded trials with excellent sensitivity and specificity [35,40]. PBMC find more reactivity to the islet cell proteins has also been demonstrated to have clinical relevance in identifying autoimmune diabetes patients with more severe loss of beta-cell function [41]. PBMCs from patients with T1D respond to between four and 18 molecular weight regions containing islet proteins, whereas normal control subjects respond to between zero and three molecular weight regions [42]. Disadvantages. Human islets are

needed to prepare the islet antigens. Twenty ml of blood is needed per patient. The antigen specificity of the T cell responses is not defined. 1 Normal human islet cells are placed into sodium dodecyl sulphate (SDS) sample buffer, boiled and then subjected to preparative one-dimensional 10% SDS-PAGE [43]. Background.  CFSE is a non-toxic fluorescent dye that is distributed evenly between daughter cells when a cell divides [44]. This dye can be used to determine the number of cells that have proliferated, in the presence or absence Pexidartinib nmr of antigen, by flow cytometry (see Fig. 2). Advantages.  This assay is more sensitive than [3H]-thymidine incorporation and the proliferation of different lineages of cells

can be determined directly by flow cytometry, making it well suited to measuring islet antigen-specific T cell responses to autoantigens [27]. Multi-colour flow cytometry can be used to gain further information on the phenotype of the cells that have proliferated,

such as their capacity to produce cytokines after a brief stimulation with anti-CD3 mAb. Alternatively, the proliferation of different cell lineages [B cells and natural killer (NK) cells, for example] can be measured in the same sample. Finally, the CFSE-based proliferation assay can be used to isolate T cell clones [45], allowing their specificity to be determined in detail [30,31]. Disadvantages.  Each sample must be analysed individually by flow cytometry. Because of the low precursor frequency of peptide and recombinant islet protein-specific T cells their responses can be variable between replicates. Dichloromethane dehalogenase This assay measures only cells capable of proliferating in vitro. 1 Draw blood into a heparin-containing tube (note: heparin is the recommended anti-coagulant because it does not interfere with immune function). Background.  Individual HLA–T cell receptor (TCR) contacts are low-affinity interactions [46]. However, cross-linking of multiple HLA/peptide complexes increases the avidity of the interaction allowing HLA/peptide multimers, such as tetramers and pentamers, to be used to stain antigen-specific T cells [47]. HLA class I tetramers were the first to be developed [22].