A simple method for the purification of human peripheral blood antigen presenting cells (dendritic cells, monocytes/macrophages, and B lymphocytes).
An adherence method was developed that enriches for antigen presenting cells (dendritic cells, monocytes/macrophages, and B lymphocytes) from peripheral blood mononuclear cell (PBMC) preparations. This method utilizes the cells' natural adherence to polystyrene tissue culture dishes and their subsequent removal with K3EDTA after incubation at 4 degrees, with gentle pipeting. Flow cytometric analysis revealed that on average, the enrichment of CD83+ dendritic cells, CD14+ monocytes/ macrophages, and CD19+ B cells increased by 12.5 to 20, 2, and 4 fold, respectively, compared to their initial numbers present in PBMC preparations. Cell viability, determined by trypan blue exclusion, was between 90 and 98%. After the enrichment procedure, the cells could still be activated by tetanus toxoid and this was shown by flow cytometric analysis, as enhancement of class II major histocompatibility complex (MHC) (31% increase) (after antigen treatment). This is a fast and economical alternative to other established methods for the preparation of pure, functionally competent antigen presenting cells derived from peripheral blood.
Delles
Anne M
AM
Department of Dermatology, Roswell Park Cancer Institute, Buffalo, New York, USA.
Rittenhouse-Olson
Kate
K
Morgan
Janet
J
Oseroff
Allan R
AR
D000328:Adult; D000938:Antigen-Presenting Cells / Q000166:cytology*; D015703:Antigens, CD; D018941:Antigens, CD19 / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology; D001772:Blood Cell Count; D001773:Blood Cells / Q000166:cytology*; D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008264:Macrophages / Q000166:cytology; D008562:Membrane Glycoproteins / Q000032:analysis; D009000:Monocytes / Q000166:cytology; D011137:Polystyrenes; D013745:Tetanus Toxoid / Q000276:immunology; D000097802:CD83 Antigen
D015703:Antigens, CD; D018941:Antigens, CD19; D004492:Edetic Acid; D006684:HLA-DR Antigens; D007136:Immunoglobulins; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D011137:Polystyrenes; D013745:Tetanus Toxoid; D000097802:CD83 Antigen