Detection and Determination of TH17 Lymphocytes Percentage in Peripheral Blood by Flow Cytometry

Abstract

Background: CD4 and IL-17 markers can be used for separating TH17 lymphocytes from the population of CD4+ lymphocytes. The best technique for detecting TH17 cells in peripheral blood is flow cytometry in which the cells need to first be activated by phorbol 12-myristate 13-acetate (PMA) and ionomycin. Moreover, monensin is used in order to prevent interleukin 17 (IL-17) secretion out of cells. Methods: Peripheral blood mononuclear cells (PBMC) were separated from freshly isolated heparinized venous blood by centrifugation on a Ficoll-Hypaque (Lymphopreb, Sigma, USA) density gradient. Cell suspension was prepared in three concentrations and cultured with three concentrations of PMA, ionomycin and monensin (each concentration was prepared in three series). Then these were incubated for 6, 12, and 20 hours. Findings: The percentage of TH17 cells was detected by culture of 2 × 106 cells/ml of peripheral blood mononuclear cells with 150 ng/ml of phorbol 12-myristate 13-acetate, 1.5 μM of ionomycin, and 500 ng/ml of monensin and incubation at 37oC in a humidified and 5% CO2 atmosphere for 12 hours. Conclusion: Optimum procedures have to be determined based on the instruments and conditions of each laboratory.

Keywords

• H17 lymphocytes
• Ionomycin
• Phorbol myristate acetate
• Monensin