Dcf 2 7 dichlorofluorescein assay

The level of the ROS in cells was determined using the DCF (2,7-dichlorofluorescein) assay (Life Technologies, Poland). For experiments, the stock solution of carboxy-H₂DCFDA (50 µg/ml in sterile DMSO; Sigma, Poland) was established at the RT in the dark and then diluted in a cell culture medium without FBS. The cells were seeded on black 96-well plates (3 × 10⁴ cells/cm²). After washing with PBS, the reagent was added to the cell culture to a final concentration of 5 µM, and cells were incubated at 37°C in darkness for 30 min. After the incubation, the mean fluorescence of DCF in wells was measured every 30 min for 90 min total using a plate reader (Multiplate Reader EnSpire, Perkin Elmer) with excitation wavelength of 495 nm and emission wavelength of 530 nm. Intracellular ROS generation assay was evaluated directly after balloon flight (0 h) and 24 h after landing (24 h).

For the ROS production, after electroporation and electroporation combined with photodynamic therapy, cells were cultured on black 96-well plates with the flat transparent bottom (Perkin Elmer, Poland) in concentration 3 × 10⁴ per well. DCF protocol was measured after 5, 10, 30, and 60 min post-irradiation by 10 J/cm², and then after 10 min. Reactive oxygen production after photodynamic reaction (PDR) was determined using DCF (2,7-dichlorofluorescein) assay (Life Technologies, Carlsbad, CA, USA). This DCF assay is based on the application of fluorescent properties of 6-carboxy-2,7-dichlorodihydrofluorescein diacetate 2,7-dichlorofluorescein (carboxy-H₂DCFDA). For experimentations, the concentrated stock solution of carboxy-H₂DCFDA (50 µg/mL in sterile DMSO) was kept at RT in the dark conditions and was subsequently properly diluted, following the manufacturer’s protocol, in cell culture medium without FBS. Then, the incubation medium was washed out from the cells with PBS containing 6 mM glucose, and the DCF reagent was added to the cell culture to achieve a final concentration of 10 µM and was left in darkness for 30 min, with incubation at 37 °C. After this time, the fluorescence was measured, where the excitation wavelength of 495 nm and emission wavelength of 530 nm was used. ROS level was detected by a multiwell scanning spectrophotometer (EnSpire Perkin Elmer, Poland).

For the ROS production after nsPEF and nsPEF combined with calcium ions, the DCF method was used. Cells were exposed to nsPEF protocols and resuspended on black 96-well plates with a flat transparent bottom (Perkin Elmer, Krakow, Poland). DCF (2,7-dichlorofluorescein) assay (Life Technologies, Warsaw, Poland) for ROS detection was implemented after 60 min post-electroporation. This method is based on the application of fluorescent properties of 6-carboxy-2,7-dichlorodihydrofluorescein diacetate 2,7-dichlorofluorescein (carboxy-H₂DCFDA). The stock solution of carboxy-H₂DCFDA (50 µg/mL in sterile DMSO) was kept at RT in dark conditions and was afterward diluted, following the manufacturer’s protocol, in a cell culture medium without FBS. As a positive ROS control, an oxidizing agent 2, 2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich) in 100 µM concentration was used. Then, the DCF reagent was added to the cell culture to achieve a final concentration of 10 µM and was left in darkness for 30 min of incubation at 37 °C. After this time, the fluorescence was measured, where the excitation wavelength of 495 nm and emission wavelength of 530 nm was used. The ROS level was detected by a multiwell scanning spectrophotometer (EnSpire Perkin Elmer, Warsaw, Poland).