The efficient isolation of murine splenic dendritic cells and their cytochemical features.
Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.
Zarnani
Amir Hassan
AH
Department of Immunology, Faculty of Medicine, Tarbiat Modarres University, Tehran, Iran.
Moazzeni
Seyyed-Mohammad
SM
Shokri
Fazel
F
Salehnia
Mojdeh
M
Dokouhaki
Pouneh
P
Shojaeian
Jaleh
J
Jeddi-Tehrani
Mahmood
M
D000818:Animals; D017951:Antigen Presentation; D039521:CD11c Antigen / Q000276:immunology; D043182:Carboxylesterase / Q000378:metabolism*; D049109:Cell Proliferation; D002469:Cell Separation; D003713:Dendritic Cells / Q000166:cytology* / Q000201:enzymology / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000502:physiology; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000201:enzymology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology; D009195:Peroxidase / Q000378:metabolism*; D013154:Spleen / Q000166:cytology*
D039521:CD11c Antigen; D009195:Peroxidase; D043182:Carboxylesterase