Journal of Immunology Research / 2012 / Article / Fig 2

Review Article

The Suckling Rat as a Model for Immunonutrition Studies in Early Life

Figure 2

Pattern of maturation of the main effector lymphocyte populations in the rat small intestine during suckling. Results were estimated from data obtained from 1, 3, 5, 7, 11, 14, and 21-day-old rats. (a) Relative proportions of intraepithelial lymphocytes (IELs) in suckling and adult Lewis rats. Intestinal IEL suspensions were obtained by incubations with DTT, EDTA and medium, and subsequent purification with 44/67.5% Percoll. Immunofluorescence staining with anti-rat antibodies to CD4, CD8α, TCRαβ, TCRγδ, and NKR-P1A was then applied. Flow cytometry analysis allowed establishing the percentage of a particular IEL subset with respect to the total number of IEL [30]. (b) Relative proportions of lamina propria lymphocytes (LPLs) in suckling and adult Lewis rats. LPLs suspensions were obtained after removing IELs, by digestion with collagenase, and purified with 44/67.5% Percoll. LPL were then stained with fluorescence-conjugated antibodies to CD4, CD8α, CD45RA, and NKR-P1A. Analyses were performed by flow cytometry and cells were expressed as the percentage of positive cells with respect to total LPL [31]. (c) Number of IgM- and IgA-secreting cells (SC) in 106 cells from small intestine lamina propria in suckling and adult Lewis rats. LPL suspensions were obtained after removing IELs by digestion with collagenase-dispase. Thereafter, serial dilutions of LPL were incubated in anti-rat IgM or IgA-coated plates. Biotin-conjugated anti-rat IgA or IgM, extravidin-peroxidase and colorimetric substrate allowed enumerating spots that corresponded to each secreting cell [31].

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