Methodology article

Efficient isolation of highly purified tonsil B lymphocytes using RosetteSep with allogeneic human red blood cells

Jonathan Zuccolo , Tammy L Unruh and Julie P Deans

Department of Biochemistry and Molecular Biology, Immunology Research Group, Institute of Infection, Immunity and Inflammation, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

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BMC Immunology 2009, 10:30doi:10.1186/1471-2172-10-30

Published:	27 May 2009

Abstract

Background

Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step, using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes, whereas single cell suspensions from tonsils contain relatively few red blood cells.

Results

B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however, the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells, but not sheep red blood cells, reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells.

Conclusion

RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods.