A method for rapid isolation of highly purified human monocytes using fully automated negative cell selection (36.25)

• Published in: The Journal of immunology (1950) 2007-04, Vol.178 (1_Supplement), p.S17-S17
• Main Authors: Yuan, Ning, Guilbault, Benoit G, Milton, Graeme T, Fadum, Jodie, Damen, Jackie E, Eaves, Allen C, Thomas, Terry E, Wognum, Albertus W
• Format: Article
• Language: English
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.178.Supp.36.25

The preparation of highly purified monocytes for experimentation has traditionally been difficult, requiring multiple steps and many hours of work. We report the successful development of a rapid negative selection method that enables the preparation of highly purified monocytes from human peripheral blood using EasySep® column-free immunomagnetic cell separation technology. A cocktail of monoclonal antibodies incorporated into tetrameric antibody complexes was used to crosslink unwanted cells in the sample to EasySep® magnetic particles. The tube containing the labelled cell suspension was then placed in an EasySep® magnet for 2.5 minutes. Unlabelled cells were recovered by pouring off the cell suspension while labelled unwanted cells were held to the walls of the tube by the magnetic field. The whole procedure was completed in 30 minutes and yielded CD14+CD16− monocyte fractions that were on average 90% pure with an average recovery above 60%. Stimulation of purified monocytes with GM-CSF, IL-4 and LPS led to efficient differentiation into dendritic cells (DC) as identified by the expression of the DC markers CD1a and CD83, and the loss of the monocyte marker CD14. Differentiated DC induced robust allogeneic CD4+ T cell proliferation, confirming that the isolated monocytes were fully competent to differentiate into functional DCs. This method was also fully automated using the RoboSep® cell separator.