2 7 dichlorofluorescein diacetate dcfh

ROS production was quantified using 2′-7′-dichlorofluorescein diacetate (DCFH) (Thermo Fisher Scientific) [21 (link)]. The cells were exposed for 30 min to 20 µM DCFH solution and the emission at 529 nm of the DCFH dye was monitored using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). The results are the means of three independent experiments performed in triplicate ± SD.

10⁴ TRPM7-silencing/-expressing HUVEC/cm² were seeded in a 24-well plate and were treated with doxycycline (2 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) on the following day for an additional 24, 48 or 72 h. The cells were then trypsinized, stained with 0.4% trypan blue solution and counted using a LUNA II automated cell counter (Logos Biosystems, Dongan-gu Anyang-si, Gyeonggi-do, Republic of Korea).
Reactive oxygen species (ROS) production was quantified using 2′-7′-dichlorofluorescein diacetate (DCFH, Thermo Fisher Scientific, Waltham, MA, USA). Stable transfected TRPM7-silencing/-expressing HUVEC were cultured into black-bottomed 96-well plates (Greiner BioOne, Kremsmünster, Austria), treated with 2 µg/mL of doxycycline for 24 h and exposed for 30 min to 20 µM DCFH solution [15 ]. The emission at 529 nm of the DCFH dye was monitored using Varioskan Lux (Thermo Fisher Scientific, Waltham, MA, USA), and the results were normalized on nuclei stained with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, St. Louis, MO, USA). The results are the means of three independent experiments performed in triplicate ± standard deviation (SD).