Encyclopedia of Experiments: Immunology
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Transcript
Following pathogen exposure, macrophages undergo activation and release pro-inflammatory and anti-inflammatory cytokines, coordinating the immune system response.
To study bone marrow-derived macrophage activation in vitro, transfer an isolated mouse bone marrow cell suspension to a culture flask. Briefly incubate.
The mesenchymal cells, monocytes, and mature macrophages adhere to the flask surface while the hematopoietic progenitors remain in suspension.
Collect the media containing hematopoietic progenitors and centrifuge. Transfer the cells resuspended in macrophage colony-stimulating factor, M-CSF, media to a culture flask.
M-CSF causes the myeloid progenitor subpopulation to differentiate into macrophages, which appear branched and adhere to the flask.
Discard the non-adherent cell-containing media. Add M-CSF media, and incubate. Once the macrophages reach their fully functional state, harvest them. Centrifuge.
Plate macrophages resuspended in M-CSF media into multi-well plate wells. Following macrophage adherence to the wells, add human polyclonal immunoglobulins and bacterial lipopolysaccharide, LPS, a toxin.
Immunoglobulins bind to specific FCγ receptors, while LPS binds to Toll-like receptors on macrophages. This co-stimulation drives anti-inflammatory macrophage activation via downstream signaling cascades, causing the release of high levels of anti-inflammatory cytokines and low levels of pro-inflammatory cytokines.
Collect the cytokine-containing media for further analysis to confirm macrophage activation.
