Las x microscope software

Intracellular ROS were measured using 2′,7′-dichlorofluorescein diacetate (DCF-DA) with a fluorescence reader and fluorescence microscopy. RAW264.7 cells were seeded at 1 × 10⁵ cells/mL in a 96-well plate and cultured overnight. Cells were pre-treated with PPB and PPS and then stimulated with LPS for 24 h. After washing with 200 μL PBS, cells were incubated with 20 μM DCF-DA in serum-free media for 30 min. The dye solution was discarded and the cells were washed twice. ROS production was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence reader (Molecular Devices, CA, USA). Cellular ROS were measured by fluorescence microscopy (Leica, Wetzlar, Germany) using LAS X microscope software (Leica).

Cellular ROS production in RAW 264.7 cells was assessed by 2',7'-dichlorofluorescein diacetate (DCF-DA) using a fluorescence reader and a fluorescence microscope. The RAW 264.7 cells were seeded at 3 × 10⁵ cells/ml in 96 well plates and incubated overnight. Subsequently, these cells were treated with different PDE concentrations (25, 50, and 100 μg/ml) and cultured for 24 h. After the treatment period, they were washed twice with 200 μl of warm phosphate-buffered saline (PBS). To assess ROS production, we treated the cells with 20 μM DCF-DA in a serum-free medium, followed by incubation for 30 min in a CO₂ incubator. The dye-containing medium was completely removed, and the cells were washed twice with 200 μl of PBS. The fluorescence intensity of the DCF-DA dye, indicative of ROS production, was measured using a microplate reader (Bio-Rad Inc.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Furthermore, cellular ROS production was visualized using fluorescence microscopy (Leica, Germany) and analyzed using LAS X microscope software (Leica).

The proteins were labeled by excitation wavelength well-separated dyes of Alexa Fluor 488, mCherry, and Alexa Fluor 647, and the high-resolution images were collected by a Leica SP8 TCS STED ×3 microscope using the lightning mode with the lowest channel cross-talk setting. The colocalization was analyzed by the Leica LAS X microscope software. The whole single cell was circled as a region of interest (ROI) for colocalization analysis of optoWASP and SRSF2 or Pol II. The nucleoli where rRNA are highly expressed but without visible optoWASP expression were not included in ROIs for colocalization analysis of optoWASP and nascent RNA. The Pearson correlation coefficient (Pearson’s r) value was calculated within the ROIs.