Analysis of EPC, ROS, and Apoptosis in Mice

Blood cells and bone marrow cells were collected and prepared from mice after 6 weeks of PM or vehicle exposure for analysis of EPCs, intracellular ROS level and apoptosis following the removal of red blood cells (RBC) using RBC lysis buffer as described [10 (link),16 (link)]. For EPC analysis, CD34⁺/CD133⁺ cell population was determined using flow cytometry as described [11 (link),16 (link),30 (link)]. The antibodies of CD34⁺ AF700 and CD133⁺ APC were purchased from BioLegend (San Diego, CA, USA). Intracellular ROS level in CD34⁺/CD133⁺ cells were quantitatively measured using FITC-ROS detection reagents (Invitrogen) as described [16 (link),31 (link)]. The cells were incubated with the reagent at 37 °C for 10 min. After 2 times of washing with PBS, the labeled cells were suspended in warm PBS and analyzed with flow cytometry. The apoptotic rate of EPCs was determined using the FITC Annexin V apoptosis detection kit from BD (Cat#556547) as per the manufacturer’s protocol. The fluorescence-positive cells were quantitatively evaluated using a Flow Cytometer LSR II (BD Bioscience, San Jose, CA, USA) and software FlowJo_V10.