Daf fm da

Measurements of the intracellular reactive oxygen (ROS) and nitrogen species (RNS) production in the VEGF‐D‐treated HUVECs were performed by monitoring the oxidation of 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCF DA) and 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF‐FM DA), respectively (Thermo Scientific, Waltham, MA, USA). Assays were performed in the 96‐well microplates, in a HBSS solution containing 5.5 mM glucose, pH 7.4. After 12 hrs of HUVEC starvation, VEGF‐D was added for 4, 8, 12 and 24 hrs. The experimental medium was then replaced to HBSS with 5‐μM probe, and the fluorescence was monitored for 1 hr using Fluoroskan Ascent FL microplate reader.

Peritoneal exudate cells were obtained from the ascites liquid. A 50-μL aliquot was taken from the sample and centrifuged at 3000× g for 5 min, then the supernatant was removed and 10 µL of the working solution of the 4-amino-5-methylamino-2’, 7’-difluorofluorescein diacetate (DAF-FM DA) (Thermo Scientific, Eugene, OR, USA) fluorescent probe was deposited at a concentration of 5 µM, and incubated at 37 °C for 30 min in the dark. Then, 500 μL of sterile PBS were added and centrifuged at 3000× g for 5 min, the supernatant was discarded, 500 µL of PBS were added, and it was incubated at 37 °C for 15 min in the dark. It was then centrifuged at 3000× g for 5 min, and the supernatant was discarded [18 (link)]. The cell packet labeled with the DAF-FM DA probe was prepared for peritoneal macrophage labeling. In total, 4 µL of the anti-F4/80 monoclonal antibody labeled with APC (Biolegend, San Diego, CA, USA) was deposited and incubated for 30 min at ambient temperature in darkness. Subsequently, 500 μL of FACSFlow was added, centrifuged at 3000× g for 5 min, and the supernatant was removed. The cell packet was resuspended in 200 μL of FACSFlow to be evaluated in the FACS Canto II (BD) flow cytometer.