Cd133 apc

For the analysis of cell-surface markers, a total of 500,000 cells/ml were stained with 0.5 ml fluorescence-activated cell sorting (FACS) buffer in Dulbecco’s phosphate-buffered saline (PBS) with 1% fetal bovine serum (FBS) for 30 min at 4 °C. Antibodies against CD133-APC (Biolegend, 372805, San Diego, CA, USA) and CD44-FITC (Biolegend, 338803, San Diego, CA, USA) were used for the flow cytometry analysis and sorting. Stained cells were analyzed using the SH800 Cell Sorter (Sony Biotechnology Inc., Tokyo, Japan) equipped with SH800 software.

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.