Isolation of Lymphocytes from a Mouse Peyer's Patch
Transcripción
Peyer's patches, PPs, aggregates of organized lymphoid tissues in the small intestinal wall, contain immune cells including B lymphocytes, T lymphocytes, dendritic cells, and macrophages.
To isolate lymphocytes from mouse PPs, begin with a euthanized mouse. Incise the abdominal wall, exposing the peritoneal cavity. Locate the cecum. Dissect the ileocecal junction to separate the cecum from the small intestine.
Excise the mesentery and incise the small intestine at the duodenum to remove the small intestine from the peritoneal cavity. Place the small intestine in a multi-well plate containing chilled medium and rinse to remove debris.
Position the small intestine with the mesenteric side facing down, and moisten with medium to maintain tissue hydration. Identify the PPs — white multi-lobulated aggregates on the anti-mesenteric side of the intestinal wall.
Carefully excise the PPs, excluding the surrounding intestinal tissue. Transfer to a tube containing medium. Incubate under constant agitation. This agitation step detaches the mucus, cell debris, and any fecal matter from the PPs, thus improving the recovery of PP lymphocytes.
Next, transfer the tissue into a cell strainer with an appropriate mesh size. Gently disrupt the PPs and release cells. Flush with medium through the strainer to obtain a single-cell lymphocyte suspension. Centrifuge the suspension at a low temperature to maintain the cell stability.
Resuspend the lymphocytes in medium, and use for further analysis.
