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OMIP-003

Phenotypic analysis of human memory B cells

Chungwen Wei, John Jung, Inaki Sanz Cytometry PART A, Volume 79A, Issue 11, 894-896 (2011)

PURPOSE: This panel was developed to characterize the phenotypic diversity of human memory B cells, with an emphasis on discriminating cell subsets within both the conventional memory population (CD27+) and the more recently described isotype switched (IgD−) population lacking expression of CD27 (1). It has been tested on fresh and cryopreserved peripheral blood mononuclear cells (PBMC), as well as bone marrow aspirates and tonsillar cells (Table 1). The multicolor panel described herein has been used extensively to analyze large numbers of PBMC samples obtained from healthy controls in steady state and in response to infection (HIV, influenza, respiratory syncytical virus) and vaccination (influenza, tetanus) as well as in hundreds of patients with autoimmune diseases (systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, psoriatic arthritis and Type 1 diabetes) and conditions characterized by allogeneic immune responses (renal transplantion and chronic graft versus host disease). This panel is also being applied in a longitudinal study in which 150 SLE patients are to be followed quarterly for a period of 2 years.

CELL TYPE: Fresh or cryopreserved PBMC, bone marrow, and tonsil mononuclear cells

MACHINE: BD LSR2

Marker Target Clone Fluorochrome Purpose
IgD Human IA6-2 FITC differentiation
CD183 Human 1C6/CXCR3 PE homing
CD24 Human SN3 PE-Alexa 610 differentiation, epithelial adhesion
CD21 Human B-ly4 PE-Cy5 activation, BCR co-receptor for antigen bound C3d
CD38 Human HIT2 PerCP-Cy5.5 differentiation
CD45R (B220) Human PE-Cy7 differentiation
CD3 Human SP34-2 Pacific Blue exclusion
Dead cells Human Live/Dead Fix Aqua exclusion
CD27 Human CLB-27/1 Qdot 605 differentiation, receptor for CD70 on activated T cell
CD95 Human Dx2 APC activation, pro-apoptotic in the presence of activated CD95L T cells
Biotin Human Streptavidin Alexa Fluor 680 n/a
CD19 Human SJ25C1 APC-Cy7 lineage