A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity
Transcript
Mast cells — white blood cells containing cytoplasmic granules filled with inflammatory mediators — play a major role in innate and adaptive immune responses.
To isolate connective tissue-type mast cells from the peritoneal cavity — a fluid-filled space harboring abdominal organs — take a euthanized mouse fixed in a supine position. Make a longitudinal incision up to the sternum, exposing the abdominal wall.
The peritoneal cavity lies underneath the abdominal wall, containing fluid that harbors several immune cells including macrophages, lymphocytes, polymorphonuclear leukocytes, and mast cells.
To isolate the cells by peritoneal lavage — aspirating the intraperitoneal contents — inject an ice-cold growth medium into the peritoneal cavity without perforating the organs, avoiding blood leakage. The cold medium helps maintain cell viability.
Carefully shake the mouse for an adequate duration, mixing the immune cells with the medium. Insert an empty syringe into the cavity. Slowly aspirate the medium — recovering most of the injected fluid for better cellular yield.
Transfer the cell suspension to a collection tube. Centrifuge to precipitate the cells as a pellet. Discard the supernatant. Resuspend the pellet in an appropriate medium.
Plate the cells and incubate them at physiological conditions, allowing the cells to adhere to the culture vessel. Add specific cytokines that bind to their receptors on the mast cells, inducing cell proliferation.
The proliferated cells are ready for downstream assays.
