Application of the ILISPOT-IDIP system for the enumeration of different sizes of IgA spot forming cells in the murine small and large intestine

Abstract The immunofluorescence-linked immunospot (ILISPOT) assay associated with the immunofluorescence digital image processing (IDIP) system was originally developed in our laboratory to allow enumeration of immunoglobulin (Ig) producing, spot forming cells (SFC) in a more objective and quantitative manner. In this study, the ILISPOT-IDIP system was further advanced in order to analyze different sizes of SFC (e.g., IgA producing cells) including large (L), medium (M), and small (S) cells which correspond to high (> 2.4 pg), medium (1.2–2.4 pg) and low ( 5 SFC/10 6 cells) when compared with large intestine (∼ 1.3 × 10 5 SFC/10 6 cells). Among the 3 areas of small intestine, the upper (∼ 3.7 × 10 5 SFC/10 6 cells) and middle (∼ 2.4 × 10 5 SFC/10 6 cells) parts had higher numbers of IgA SFC when compared to the lower small (∼ 2.3 × 10 5 SFC/10 6 cells) intestine. When these IgA producing cells in different parts of the intestine were classified into three groups according to the size of individual spots, the upper and middle intestine contained higher frequencies of large (∼ 20%) and medium (∼ 20%) SFC which corresponded to high and medium IgA secretors in comparison to the lower small (∼ 9%) and large (∼ 6%) intestine. In contrast, the lower small and large intestine were dominated by small SFC since ∼ 85% of IgA producing cells were categorized as low secretors. Using the advanced ILISPOT-IDIP system, a unique distribution of different sizes (or secretion rates) of IgA producing SFC was elucidated in the different regions of the mouse small and large intestine.