Lsr flow cytometry

MCF-7, T47D and MDA-MB-175 cells were treated with Thiolutin for cell cycle analysis. For siPSMD14 knockdown experiments, MCF-7 cells were transfected with siPSMD14 or siControl. Similarly, for MCF-7 Y537S cells, they were treated with siPSMD14 or Tamoxifen. After 24 h, the treated cells were digested and resuspended into a single cell suspension, followed by washing in PBS. Ethanol was used to fix the cells, which were subsequently stained with propidium iodide. Fluorescence intensity was measured using BD LSR flow cytometry.

To detect mitochondria activity of fresh TAMs, we first collected leukocyte mononuclear cells from peritoneal elute cells by density gradient centrifuge. Then, we stained leukocyte mononuclear cells with macrophage surface antibodies for 30 minutes. Following this, cells were washed and stained with mitochondria reagents (Life Technologies, Thermo Fisher Scientific) for 30 minutes at 37°C in RPMI-1640 without FBS. MitoTracker Green (100 nM) and MitoTracker Deep red (100 nM) were combined to detect the mitochondria mass. MitoSOX (5 μM) was used to check mitochondria-related ROS. To detected the effect of arginine on the induced mitophagy, TAMs were treated with oligomycin (10 μM) and antimycin A (4 μM) (control) in the presence or absence of rapamycin (100 nM). Meanwhile, arginine or arginanse-1 inhibitor nor-NOHA (0.5 mM) was added into the culture for 24 hours. The levels of damaged mitochondria without any inhibitors were used as background control. The percentages of accumulated damaged mitochondria were normalized to the control group. After washing with PBS for 2 times, the stained TAMs were gated and detected via BD LSR flow cytometry.