The density of PMN is carefully selected to achieve a clear separation of mononuclear and polymorphonuclear cells after centrifugation through the media. Whole uncoagulated, fresh blood is layered over a nearly equal volume of PMN in a centrifuge tube and spun at 500xg for 30 minutes. Mononuclear cells appear as a discrete layer at the media interface while neutrophils form a second band within the media above the pelleted red blood cells. The neutrophil layer is collected by pipet and diluted in saline or culture media before further analysis. PMN is provided as a sterile, pyrogen-free solution in 100 ml bottles and comes with complete instructions.
Unopened bottles are stable at room temperature when stored out of direct light.
Red-Out contains a murine monoclonal antibody to a cell surface antigen found on all human erythropoietic cells. When mixed with whole blood, it causes small aggregates of red blood cells to form that quickly sediment through separation media. It is particularly useful on samples that contain a high percentage of immature, nucleated erythrocytes that co-sediment and contaminate lymphocyte preparations. Red-Out treatment of fetal blood and blood from immunosupressed patients quantitatively removes red blood cells yielding remarkably clean lymphocytes. Red cell aggregates that have formed sediment to the bottom of the tube during centrifugation.