Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Transcript
Peritoneal macrophages, residing in the peritoneal cavity engulf pathogens and secrete pro-inflammatory and anti-inflammatory cytokines to modulate the immune response.
To study in vivo activation of peritoneal macrophages, begin by restraining the mouse to minimize its movement. Take a mixture of lipopolysaccharides — pathogen-derived antigens and intravenous immunoglobulins and inject them intraperitoneally.
Post-injection, lipopolysaccharides bind to macrophage's pattern-recognition receptors, triggering an immune response and releasing pro-inflammatory cytokines, including IL-12.
In contrast, immunoglobulins interact with macrophage Fc receptors, downregulating the immune response by suppressing IL-12 production and promoting the release of anti-inflammatory cytokines, including IL-10. This co-stimulation by lipopolysaccharides and immunoglobulins activates peritoneal macrophages into anti-inflammatory macrophages.
Secure the euthanized mouse and make a longitudinal incision along the abdominal cavity. Retract the skin from the abdominal muscle. Inject a buffer into the peritoneal cavity; agitate to dislodge cells from the peritoneal cavity.
Collect the activated macrophage-containing peritoneal fluid.
Culture these activated macrophages in a medium containing co-stimulatory molecules — immunoglobulins, and lipopolysaccharides to enable cytokine production. Collect the spent medium and quantify cytokine levels using an enzyme-linked immunosorbent assay.
A higher IL-10 concentration than IL-12 confirms successful peritoneal anti-inflammatory macrophage activation.
