A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation

Abstract

The standard isolation procedure for antigen presenting dendritic cells (DC) takes 2 days and includes selective adherence to tissue culture plates which may lead to the activation of these cells. This report describes the isolation of DC by centrifugal elutriation (CE). Murine spleen cells were separated on the basis of size and density into 7 CE fractions. This method took 90 min. Ceils from each CE fraction were characterized by fluorescence activated cell sorter (FACS) analysis and their antigen presenting cell (APC) activity was determined by a secondary Sendai virus specific T cell proliferation assay. CE fraction 5 contained most of the DC with a concentration of 6-10% , representing an approximately 15-fold enrichment compared to unseparated spleen cells ( < 1 % DC). This CE fraction also exhibited the highest APC activity, which was almost completely abolished after depletion of DC by treatment with monoclonal antibody 33D1 (DC-marker) and complement. Further enrichment of CE fraction 5 by discontinuous density gradient centrifugation resulted in a cell population containing 35-55% 33Dl-positive cells with similar characteristics as DC isolated by the standard procedure, such as the capacity to induce a primary viral peptide specific CTL response. Two-color FACS analysis showed an increase in MHC expression on 33Dl-positive cells of CE fraction 5 after 18 h culture involving cell adhesion to a similar level as the M HC expression on DC isolated by the standard procedure. During this same period their morphology changed from a round to a dendritic appearance. In conclusion, our results indicate that CE is well suited for isolating DC more rapidly and without activation of these cells by adherence, a process which readily occurs in the standard isolation procedure.

Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells.

“…These data agree with those obtained from LC, studied either in situ, shortly after isolation, or after short-term in vitro culture (15,(44)(45)(46). Similarly, culture of mouse DC leads to an increase in MHC class II expression and remodeling from a rounded morphology to one in which long cytoplasmic processes are present (47,48). Of note, MHC class II localization shifted from intracellular to the cell surface upon culturing.…”

Clinical-scale elutriation as a means of enriching antigen-presenting cells and manipulating alloreactivity

“…Through subsequent immunomagnetic separation, using the same volume of selection reagents, DC could be purified in larger numbers compared with the apheresis product. Secondly, it was found that effector memory CD4 ' T cells were enriched and naive CD4 ' T cells depleted in Elutra F3 and F4, and this correlated with relatively The finding of DC enrichment in Elutra fractions is not necessarily unexpected as it has been described previously in two studies with non-clinical elutriation [12,14]. However, it has not been demonstrated previously using the Elutra system [10,15Á24], which differs from these nonclinical studies in several ways.…”

Rapid flow cytometric identification of putative CD14? and CD64? dendritic cells in whole blood

“…In addition, no surface antigens are specific for this population of cells (27). These problems were partially circumvented by the development of isolation procedures (28,31,34,39,41), which relied on the negative selection of DCs based on their lack of the cell surface antigens, such as CD2, CD3, CD14, CD16, CD19, CD20, CD56, and CD57, which are found on T lymphocytes, monocytes, neutrophils, B lymphocytes, and natural killer (NK) cells, respectively. Unfortunately, these procedures took 18-36 h and included several stages, one of which involved the selective adherence to tissue culture plates, which could lead to cell activation (14,16,32).…”