Enrichment of PBMCs with Monocytes Protocol Protocol for enrichment of PBMCs with monocytes. The cell suspension obtained after this protocol contains 40-70% monocytes. This cell suspension is than used for positive or negative MACS separation.
Isolation and Culture of Mouse Bone Marrow-Derived Macrophages Protocol Mononuclear phagocyte progenitor cells derived from femoral and tibial bone marrow are propagated in the presence of M-CSF. This macrophage growth factor is secreted by L929 cells and is used in the form of L929 cell conditioned medium. The progenitor cells proliferate and differentiate through monoblast, promonocyte and monocyte stages before maturing to macrophages. At this time the cells become firmly adherent to the culture vessel.
Isolation of Dendritic Cells Protocol Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells.
Isolation of Monocytes from Enriched PBMCs using CD14 Magnetic Beads Protocol This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment.
