Improved Isolation Methods for Mucosal Leukocytes from Small and Large Intestines in Rats

Abstract

We optimized the isolation protocol for intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from the rat small intestine, and LPLs from even the rat large intestine. The major population of IELs in the small intestine was considered to be from the villus epithelia. The cytotoxicity of mucosal leukocytes was comparable among isolated fractions from both the small and large intestines, regardless of the population differences. Further analyses of the cells collected from other lymphoid tissues demonstrated that CD161(+) cells selectively accumulated in the intestinal lamina propria and did not recirculate through the lymph ducts. Our modified isolation protocol enables the collection of mucosal immune cells from the rat intestines without any deterioration of cell function and could contribute to a better understanding of dietary influences on the mucosal immune system.

Mucosal permeability is an intrinsic factor in susceptibility to dextran sulfate sodium-induced colitis in rats

“…28 Cytokine production from isolated mesenteric leukocytes Leukocytes were isolated from mesenteric lymph nodes, as previously described. 30 The viability of mesenteric leukocytes (MLs) was assessed immediately after their isolation by trypan blue exclusion. The MLs (1.0 Â 10 6 cells/well) were stimulated with various concentrations of DSS (0.04, 0.4, 2 or 4% wt/vol as final concentrations) in a 48-well plate for 24 h, after which the media, including the cells, were collected.…”

Upregulation of 11β-hydroxysteroid dehydrogenase 1 in lymphoid organs during inflammation in the rat

“…The colon, draining MLN, and spleen were harvested and used immediately for the measurement of enzyme activity and isolation of distinct cell types. Intraepithelial (IEL) or lamina propria leukocytes (LPLs) were isolated as described previously [19,23]. Splenic and MLN cell suspensions were prepared in RPMI 1640 medium by pressing the organs through nylon gauze (mesh size 45 m).…”

Depletion of CD8α+ lymphocytes attenuates CCL28 expression in villus epithelia in rats

“…Additionally, this finding indicates that CD8␣␣ + IELs contribute to CCL28 expression in the villus epithelia. Considering our previous flow cytometric analysis of IELs and LPLs [26], CD8␣␣ + IELs probably express CD45RA and ␥␦ T cell receptor. A CD8␣ depletion study, using a specific antibody, would be useful for understanding the mechanism of the IEL differentiation pathway in the intestinal mucosa.…”

“…Lymphocyte isolation from peripheral blood, mesenteric lymph nodes, and small intestine without Peyer's patches was performed as described previously [26]. Isolated cells (1.0 × 10 6 cells) were stained to evaluate the CD8␣ cell phenotype by flow cytometry.…”