Detection of subsets of CD3+CD20+ T cells in healthy subjects. (A) (i) Typical flow cytometry dot plots of the forward side scatter (FSC) against sideward scatter (SSC) of peripheral blood lymphocytes (PBLs). Within the lymphocyte population, CD3-FITC/CD19-APC/CD20-PE triple-staining was performed on the cells. This staining enabled visualization of (ii) double-positive CD3+CD20+, (iii) CD3+CD19-, and (iv) CD19+CD20+ events. (B) PBLs expressing a CD3+ phenotype (α-CD3-FITC) were gated and evaluated for their expression patterns of CD19 (α-CD19-APC) and CD20 (α-CD20-PE). Alternatively, PBLs were stained with α-CD4-FITC or αCD8-FITC and α-CD19-APC/α-CD20-PE to determine the percentage of CD4+/CD20+/CD19- and CD8+/CD20+/CD19- cells. (C) Lymphocytes were stained with α-CD3-Pacific blue/α-CD19-APC/α-CD20-FITC, whereupon α-CD3+/α-CD19-/α-CD20+ and α-CD3+/α-CD19-/α-CD20- cells were sorted as illustrated with the sort plots. Subsequently, sorted populations were stained with a mix of α-CD4-PE/α-CD8-PE and the respective populations were analyzed by confocal microscopy for CD4/CD8 (red pseudo color) and CD20 (green pseudo color). Confocal microscopy was performed with a Leica DM IRE2 Inverted microscope (objectives: HCX PL APO 63 ×/1.3 with glycerin; camera: Stanford Photonics XR/Mega-10I (intensified) charge-coupled device (CCD) camera; software: Yokogawa Confocal Scanner Unit CSU10). This experiment was performed on four occasions with similar results. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
Eggleton et al. Arthritis Research & Therapy 2011 13:R208 doi:10.1186/ar3541