Eclipse ti e

BV2 cells were inoculated in a six-well plate for 12 h and then Mock and HSV-1 infection. After 24 h of infection, pyroptotic and apoptotic cell death were evaluated with PI staining (2 μl/mL)/Hoechst 33342 (5 μl/mL) and Annexin V-APC (6 μl/mL)/Hoechst33342 (5 μl/mL) (Zha et al., 2016 (link)). PI staining (#638) and Hoechst 33342 (#639) were purchased from immunochemistry. Annexin V-APC was purchased from KGI Biosciences. Dead cells (Annexin V-APC and PI permeable) were determined under a Nikon light microscope (Eclipse Ti-E) and analyzed with NIS-Elements Viewer 4.50 and ImageJ program.

Indicator for nitric oxide determination: 4,5-diaminofluorescein diacetate (DAF-FM diacetate, Molecular Probes). The non-fluorescent dye is pass across cell membrane. The cell’s estherase activity turns the non fluorescent dye into a weakly fluorescent form and nitric oxide binds to this intracellular dye and increases the fluorescence activity in cells. The effect of NO inhibitors (7-NI and L-NAME) on NO production in the SH-SY5Y cell model was detected using an inverted fluorescence microscope (NIKON Eclipse Ti-E) and the NIS Elements AR program. The neuroblastoma cells of the SH-SY5Y line containing PgP and MRP proteins were seeded into 24-well plates, with 6 × 10⁴ cells per well and incubated for 3 days. After seeding, the cells were treated with NO inhibitors L- NAME (100uM) and 7-NI (50uM) during 24 h. After treatment, the cells were stained with 10uM DAF-FM diacetate (30 min in 37 °C in CO₂ incubator), rewashed by DMEM buffer (without FBS and phenol red) and measured by fluorescence miscroscope exc/em 488/510 nm. Image J program was used for image analysis.