Evaluation of methods using adherence to substrate and density gradient for the isolation of human monocytes.
A new method for monocyte isolation based on cell adherence to gelatin-coated plastic and dislodgement of the adhering monocytes at low temperature was compared with 5 other methods based on cell adherence to substrate and density gradient separation. All methods produced yields of monocytes ranging approximately between 50-70% with about the same degree of purity (less than 90%) except for the method using Percoll density gradient centrifugation where the purity of monocytes was about 80%. When lidocaine at different concentrations was used for cell dislodgement or Percoll density gradient for separation, phagocytosis, Fc receptor function and cytotoxicity were adversely affected, unlike in methods using EDTA or low temperature for dislodgement of the adherent cells. In monocyte chemotaxis assays the rate of migration was affected but not the number of migrating cells for all the isolation procedures investigated. Cell spreading function was apparently well maintained only when gelatin coated plastic was used for adherence and low temperature for cell dislodgement. These data indicate that the newly described method, similar to methods using EDTA for cell dislodgement, yielded relatively intact monocytes but unlike the latter method with better preserved cell spreading. Thus, this method can be considered for standardization to obtain pure monocyte populations from peripheral blood which then can be submitted for comprehensive biochemical and physiologic studies.
D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D002634:Chemotaxis, Leukocyte; D005780:Gelatin; D009000:Monocytes / Q000502:physiology*; D010587:Phagocytosis
D005780:Gelatin