Optimizing follicular dendritic cell isolation by discontinuous gradient centrifugation and use of the magnetic cell sorter (MACS)

Abstract

Follicular dendritic cells (FDC) contribute minimally to the total cell population of lymphatic tissue. In order to obtain higher numbers of viable FDC with only a small fraction of contaminating cells the following procedure was developed. Subsequent to the usual mechanical and enzymatical digestion of human tonsils, single cells were layered on top of a discontinuous bovine albumin gradient and centrifuged at 8500 x g. The suspension collected from the 1.052-1.030 interphase contained an average of 10.5% FDC. Next, the preparation was subjected to a new step involving separation of FDC previously treated with biotin-labelled KiM4 monoclonal antibody, raised against FDC, and attached via biotin-streptavidin bonding to streptavidin-conjugated paramagnetic beads. Purification on a magnetic cell sorter (MACS) yielded 3.3-10.1 x 10(6) cells with an average FDC content of 78.4%. The viability and morphology of the resulting FDC population was examined using trypan blue staining or electron microscopy. This technique will permit in vitro studies and long term cultures with FDC isolated from human lymphatic tissue.