Intracellular ROS Assessment in PBMC

Intracellular ROS generation in PBMC cells (5 × 10⁵ pelleted cells) was measured by flow cytometric assay method [13 (link)] using a ROS-sensitive cell-permeable dye 2′ 7′ dihydrodichlorofluorescein diacetate (2′ 7′ H2DCFDA). In this method, 2′ 7′ H₂DCFDA oxidized to highly fluorescent 2′ 7′-dichlorofluorescein (2′ 7′ DCF) presence of ROS in the PBMC. The PBMC exhibited an increased fluorescence of oxidized DCF, as measured by a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA) fitted with an argon-ion laser (15 mW) set to a wavelength of 488 nm. The fluorescence of DCF was collected in FL1 channel, equipped with a 530/30 nm band-pass filter. Fluorescence was measured in the long mode using “Cell Quest Pro” software (BD Bioscience, San Jose, CA) and expressed as geometrical mean fluorescence channel (GMFC). Cells were gated on the basis of their characteristic morphology, i.e., forward scatter and side scatter of monocytes and lymphocytes. Acquisitions were performed on 10000 gated events; while data analysis was carried out with “Cell Quest Pro” software (BD Bioscience).