Expression of the S1P1 receptor in circulating immunocompetent cells by flow cytometry

The differential expression of S1P₁ receptor was measured with flow cytometry in leukocyte populations (T cells, B cells and monocytes) by using mAbs capable of recognizing specific cell markers (CD4⁺, CD8⁺, CD19⁺ and CD14⁺, respectively) conjugated with different fluorochromes. In particular, 1 × 10⁶ PBMC in PBS pH 7.4 from each patient or NHS, were labelled with allophycocyanin (APC) -anti-human CD4 (clone: OKT4, 0.6 μg/ml), peridinin-chlorophyll-protein-cyanine5.5 (PerCP-Cy5.5) anti-human CD8 (clone: RPA-T8, 2.5 μg/ml), APC-anti-human CD19 (clone: HIB19, 1.25 μg/ml) and PerCP-Cy5.5-anti-human CD14 (clone: 61D3, 5 μg/ml) mAbs, (all from Affymetrix, eBioscience). After incubation for 15′ at room temperature in dark, cells were then fixed with 1% paraformaldehyde in PBS pH 7,4 and after washing, permeabilized with 0,02% saponin-PBS-1% FCS and labelled with phycoerythrin (PE)- anti-human S1P1/EDG-1 mAb (clone: 218713, 10 µl, R&D Systems). As control isotypes, mouse IgG2bk PE-conjugated (clone:A-1, MyBiosource), mouse IgG1k- PerCP-Cy5.5 -conjugated (clone: p3.6.2.8.1) and mouse IgG2bk-APC-conjugated (clone: eBMG2b) (all from Affymetrics, eBioscience) were used. Cells were analyzed on a FACSCalibur flow cytometer using Cell Quest Pro software (BD Biosciences). The extent of expression was assessed as Specific Fluorescence Index (SFI) calculated as ratio of the mean fluorescence values obtained with the specific anti-S1P₁ as well as anti-CD markers mAbs to the respective isotype control antibody³².