Flow cytometry was performed on a FACSCanto II flow cytometer (BD Biosciences, USA).
Briefly, 50 μl of fresh whole blood was incubated with the appropriate amounts of fluorochrome-labelled monoclonal antibodies CD45 Krome Orange (clone J33), CD4 FITC (clone 13B8.2), CD25 APC (clone IHT44H3) and CD39 PC5.5 (clone BA54) from Beckman Coulter, France. Incubation was done at room temperature in the dark for 15 min using appropriate mouse immunoglobulin isotypes as a control. Following incubation, 1 ml erythrocyte lysing solution (VersaLyse, Beckman Coulter) was added to the samples and incubated under the same conditions for 20 min. Then, cells were fixed and permeabilized (using intraprep permeabilization reagent, Beckman Coulter), followed by intracellular staining with anti-FoxP3-PE (clone 259D, Beckman Coulter, France) for 30 min. Cells were resuspended in PBS and analysed. Finally, the cells were characterized by flow cytometry analysis using BD FACEDiva Software. Analysis performed with CD4 + CD25 + CD39+ FOXP3+ T-reg cells expressed as a percentage of the whole CD4 subset (Fig.1 ).![]()
Briefly, 50 μl of fresh whole blood was incubated with the appropriate amounts of fluorochrome-labelled monoclonal antibodies CD45 Krome Orange (clone J33), CD4 FITC (clone 13B8.2), CD25 APC (clone IHT44H3) and CD39 PC5.5 (clone BA54) from Beckman Coulter, France. Incubation was done at room temperature in the dark for 15 min using appropriate mouse immunoglobulin isotypes as a control. Following incubation, 1 ml erythrocyte lysing solution (VersaLyse, Beckman Coulter) was added to the samples and incubated under the same conditions for 20 min. Then, cells were fixed and permeabilized (using intraprep permeabilization reagent, Beckman Coulter), followed by intracellular staining with anti-FoxP3-PE (clone 259D, Beckman Coulter, France) for 30 min. Cells were resuspended in PBS and analysed. Finally, the cells were characterized by flow cytometry analysis using BD FACEDiva Software. Analysis performed with CD4 + CD25 + CD39+ FOXP3+ T-reg cells expressed as a percentage of the whole CD4 subset (Fig.
A representative flowcytometric plot demonstrating FoxP3 versus CD25 and CD39 expression on CD4+ T cells