Confocal sp8 smd microscope

KUP5 or Hepa 1-6 cells were exposed to nanoparticles at 37 °C and 5% CO₂. After 3 h exposure to nanoparticles, the cells were washed twice with PBS and treated with 5 μM of MitoSOX (Invitrogen, Carlsbad, CA) in HBSS at 37 °C and 5% CO₂ for 20 min. The cells were fixed with 4 % paraformaldehyde in PBS, stained with Hoechst 33342 (5 μg/mL), and imaged using a Leica Confocal SP8-SMD microscope.

KUP5 or Hepa 1-6 cells were cultured in an 8-well Lab-Tek chamber slide at 5 × 10⁴ cells/400 μL medium at 37 °C and 5% CO₂. The KUP5 cells were primed with LPS (1 μg/mL) for 4 h and incubated with nanoparticles at 50 μg/mL for 3 h. The treated cells were washed with PBS and stained with FAM-FLICA Caspase-1 or Caspase 3/7 substrates for 1 h at 37 °C according to the manufacturer’s protocol. Finally, the cells were fixed with 4% paraformaldehyde in PBS, stained with Hoechst 33342 (5 μg/mL), and imaged using a Leica Confocal SP8-SMD microscope.

KUP5 cells were treated with GOs for 16 h, then the cells were washed three times with PBS and treated with 5 μM MitoSOX in HBSS at 37 °C for 10 min. The cells were fixed with 4 % paraformaldehyde in PBS, stained with Hoechst 33342, and imaged using a Leica Confocal SP8-SMD microscope. The fluorescence intensity was monitored as the rate of oxidation of the dye in the cells at excitation/emission wavelengths of 510/580 nm. Before GO exposure, KUP5 cells were treated with various inhibitors, including a calcium chelator BAPTA-AM, a PLC inhibitor U-73122, an NADPH oxidase inhibitor DPI, and a phagocytosis inhibitor WM, for comparison purposes.

After KUP5 cells were cultured overnight in an 8-well Lab-Tek chamber slide at 5 × 10⁴ cells per well, the cells were primed with LPS (1 μg/mL) for 4 h and exposed to particles (50 μg/mL) for 1 h at 37 °C. The cells were washed with PBS and incubated with Magic Red working solution for 30 min at 37 °C. Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS. Finally, the cells were stained with Hoechst 33342 (5 μg/mL), washed with PBS, and imaged under a Leica Confocal SP8-SMD microscope.

KUP5 cells were cultured in 8-well Lab-Tek chamber slide and loaded with the plasma membrane permeable calcium indicator Fluo-4 AM ester (5 μM) in a Pluronic F-127-buffered DMSO solution for 1 h at 37 °C. Then, cells were washed in an indicator-free medium to remove any dye that is nonspecifically associated with the cell surface, followed by the incubation for a further 0.5 h to allow complete de-esterification of intracellular AM esters. Finally, the cells were fixed with 4 % paraformaldehyde in PBS, stained with Hoechst 33342, and imaged using a Leica Confocal SP8-SMD microscope. The fluorescence intensity was monitored at excitation/emission wavelengths of 494/516 nm. To confirm the role of calcium flux, KUP5 treated with a calcium chelator BAPTA-AM (10 μM) or other inhibitors, including a PLC inhibitor U-73122, an NADPH oxidase inhibitor DPI, and a phagocytosis inhibitor WM, for comparison purposes.

The KUP5 cells, primed with LPS (1 μg/mL) for 4 h, were incubated with 25 μg/mL particles, followed by washing in PBS and staining with FAM‐FLICA caspase‐1 substrate for 1 h at 37 °C. The cells were stained with Hoechst 33342 for 15 min and imaged using a Leica Confocal SP8-SMD microscope. The quantification for fluorescence intensity in the cells was monitored at excitation/emission wavelengths of 492/520 nm by a microplate reader. Treatment with Gd₂O₃ nanoparticles was used as a positive control that induces lysosomal damage.^{[36 (link)]}

KUP5, LSEC, and Hepa 1–6 cells, seeded at 2 × 10⁵ cells/well in an 8-well Lab-Tek chamber slide, were incubated with 25 μg/mL of BN and MoS₂, respectively. The treated cells were washed in PBS and stained with FAM-FLICA Caspases 3/7 substrates at 37 °C for 1 h according to the manufacturer’s instructions. Finally, the cells were stained with Hoechst 33342 for 15 min and imaged using a Leica Confocal SP8-SMD microscope. The quantification for fluorescence intensity in the cells was monitored at excitation/emission wavelengths of 492/520 nm by a microplate reader. ZnO nanoparticles were used as a positive control.