Ros probe

The cryopreserved tube contianing 1.5 mL cells was taken out from the liquid nitrogen tank and quickly placed in water bath at 37 °C for about 2 min. The cell suspension in the tube was moved into a 15 mL centrifuge tube, to which five mL complete medium was added, and centrifugal at 300 ×g for 5 min at room temperature. After removing the supernatant, cells were resuspended with a moderate amount of complete medium heavy precipitation, inoculated in a 10 cm petri dish, added with complete medium to 10 mL, and cultured again in 37 °C and 5% CO2, saturated humidity. 1 µL ROS probe (Beyotime, Shanghai, China) was added to the resuscitated cells in the proportion of 1:1,000, mixed, incubated at 37 °C for 20 min, and oscillated several times per 5min. After 5-centrifugation, cell precipitation was collected, one mL PBS was resuscitated, and centrifuged at 500 × g for 5 min. one mL PBS was resuspended to be measured. A negative control group was set up and treated in the same way without adding the probe. FITC signal was detected by flow cytometry, FITC signal was detected by FL-1A channel, PI signal was detected by FL-2A channel.

FACS analysis was performed using routine protocols with a FACS Calibur flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ, USA). Antibodies for staining included APC anti-mouse CD11b (Biolegend, San Diego, CA, USA), Alexa 488 anti-mouse F4/80 (Biolegend), biotin anti-mouse Ly6G (Biolegend) and avidin-PE (eBioscience), PE anti-mouse MR (Biolegend). Dead cells were excluded by propidium iodide staining.
For intracellular staining, cells were stained with cell surface makers first, and then washed with PBS. After centrifuge, cell pellets were suspended completely with fixation buffer on ice for 30 min, and then permeabilized with a Permeabilization Buffer (eBioscience). After centrifuge again, cell pellets were washed and stained with PE anti-iNOS (eBioscience) followed by FACS analysis.
To determine ROS production, cells were harvested and labeled with a ROS probe, according to the recommended protocol (Beyotime), followed by FACS analysis. Briefly, the culture medium was removed and the cells were incubated in dark with the redox-sensitive fluorescent dye (DCFH-DA, 10 µM) diluted by serum-free medium at 37°C/5% CO₂ incubator for 30 min. The cells were then washed with PBS and the fluorescent intensity of DCF was detected by FACS. The ROS level was analyzed using the mean fluorescent intensity (MFI).