Use of colloidal silica (Sepracell-MN) for enrichment of dendritic cells from human peripheral blood: comparison with other methods.
Three methods are described for enrichment of dendritic cells from human peripheral blood. In method 1, mononuclear cells were incubated in plastic tissue culture flasks for two h. Nonadherent cells were removed. Adherent cells were washed to remove floating cells and incubated for 14 h at 37 degrees C in 5% CO2. Carbonyl iron was added, and the flasks were incubated for another 2 h. Nonadherent cells were subjected to centrifugation over metrizamide gradient. Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells. The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively. In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low-density cells. Monocytes from this low-density cell population were removed by panning over human gamma globulin-coated plastic Petri dishes. In this method the average purity and yield of DC were 63.8% and 0.1%, respectively. In method 3, released adherent cells were treated with anti-CD5 and anti-CD14 monoclonal antibodies plus baby rabbit complement for 15 min, washed, and centrifuged with colloidal silica (Sepracell-MN). Centrifugation with Sepracell-MN was repeated three times. Low-density cells were panned twice over human gamma globulin-coated plastic Petri dishes. Nonadherent cells were highly enriched for DC. Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry. Contamination of monocytes was less than 2%. This method provided greater than 85.0% purity and 0.4% yield. This method (method 3) combines adherence, complement-dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC.
Department of Pediatrics, University of Pennsylvania School of Medicine.
J
J
Chehimi
S E
SE
Starr
H
H
Kawashima
N F
NF
Hassan
S D
SD
Douglas
S
S
Bandyopadhyay
D000906:Antibodies / Q000276:immunology; D000943:Antigens, Differentiation / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D001773:Blood Cells / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D018956:CD5 Antigens; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D002498:Centrifugation; D003102:Colloids; D003165:Complement System Proteins / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D054354:Iron Carbonyl Compounds; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology; D007962:Leukocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors; D008793:Metrizamide; D009942:Organometallic Compounds; D010641:Phenotype; D012822:Silicon Dioxide; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology; D005719:gamma-Globulins
D000906:Antibodies; D000943:Antigens, Differentiation; D015214:Antigens, Differentiation, Myelomonocytic; D018956:CD5 Antigens; D003102:Colloids; D018950:Lipopolysaccharide Receptors; D009942:Organometallic Compounds; C062165:Sepracell-MN; D005719:gamma-Globulins; D054354:Iron Carbonyl Compounds; D012822:Silicon Dioxide; D003165:Complement System Proteins; D008793:Metrizamide