Figure 1
Figure 1. Monitoring S1P1 activity in human T-cells. S1P responses were measured by flow cytometric analysis in separated PB T-cells after 18 hours of incubation in serum-free medium. (A) PB T-cell subpopulations: naïve (CD45RA+, CD27+), central memory (CM) (CD45RA−, CD27+), effector memory (EM) (CD45RA−CD27−) cells identified after permeabilization with a PE-labeled phospho-Akt monoclonal antibody or fluorescein isothiocyanate-labeled phalloidin to detect actin polymerization. Responses to SDF-1a were assessed as a positive control. (B) PB T-cells were pre-treated with 250 nM FTY720 prior to assessment of S1P-induced actin polymerization and S1P-induced Akt phosphorylation. This experiment is representative of 3. (C) PB T-cells were pre-treated with 10 uM W146 for 1 hour prior to measurement of S1P-induced Akt phosphorylation and actin polymerization. This experiment is representative of 2. Actin pol., actin polymerization.

Monitoring S1P1 activity in human T-cells. S1P responses were measured by flow cytometric analysis in separated PB T-cells after 18 hours of incubation in serum-free medium. (A) PB T-cell subpopulations: naïve (CD45RA+, CD27+), central memory (CM) (CD45RA−, CD27+), effector memory (EM) (CD45RA−CD27−) cells identified after permeabilization with a PE-labeled phospho-Akt monoclonal antibody or fluorescein isothiocyanate-labeled phalloidin to detect actin polymerization. Responses to SDF-1a were assessed as a positive control. (B) PB T-cells were pre-treated with 250 nM FTY720 prior to assessment of S1P-induced actin polymerization and S1P-induced Akt phosphorylation. This experiment is representative of 3. (C) PB T-cells were pre-treated with 10 uM W146 for 1 hour prior to measurement of S1P-induced Akt phosphorylation and actin polymerization. This experiment is representative of 2. Actin pol., actin polymerization.

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