Intracellular ROS regulates circadian oscillations of mouse and human leukocytes. (A) Blood samples were taken from 10-week-old NSG and humanized mice every 6 hours (ZT1-ZT25) (n = 6). Mouse and human CD45⁺ leukocytes from different times were separated by cell sorting and analyzed for HIF-1α expression by RT-PCR. Shown are fold changes of HIF-1α mRNA levels after normalizing data from each point in individual mice to the initial time, ZT1. (B) Blood samples were taken from points ZT7 and ZT19 in 10-week-old NSG and humanized mice (n = 6). ROS expression levels in mCD45⁺ and hCD45⁺ cells were analyzed by flow cytometry. Shown is statistical analysis of ROS MFI. (C-H) Ten-week-old NSG and humanized mice were treated with PBS and NAC (n = 4). Blood samples were taken from points ZT7 and ZT19 and analyzed for ROS (C-E) and CXCR4 staining (F-H). (I) Blood samples were taken from 10-week-old humanized mice treated with PBS or NAC (n = 6). The number counts of mCD45⁺ and hCD45⁺ cells at each point in individual mice were normalized to the initial number account of time ZT1. Shown are fold changes of cell number. *P < .05; **P < .01.