Glass bottomed dish

Mitochondrial localization of various DIL-loaded liposomal formulations in HUVECs and 4T1 cells was observed using CLSM. Cells were seeded at a density of 1×10⁴ cells/well in a glass-bottomed dish (Φ 15 mm; NEST). After 24 h of incubation, the cells were treated with RGD/DIL-Lips, KLA/DIL-Lips and RGD-KLA/DIL-Lips (DIL concentration of 2 μM) for 8 h. Subsequently, the cells were washed twice with cold PBS and stained using Mitotracker Green FM (75 nM) at 37°C for 30 min. Stained cells were rinsed three times with PBS to remove free tracking agent and then observed using CLSM.

Mitochondrial localization of various DIL-loaded liposomal formulations in HUVECs and 4T1 cells was observed using a CLSM. Cells were seeded at a density of 1×10⁴ cells/well in a glass-bottomed dish (Φ 15 mm; NEST). After 24 h of incubation, the cells were treated with RGD/DIL-Lips, KLA/DIL-Lips and RGD-KLA/DIL-Lips (DIL concentration of 2 μM) for 8 h. Subsequently, the cells were washed twice with cold PBS and stained using Mitotracker Green FM (75 nM) at 37°C for 30 min. Stained cells were rinsed three times with PBS to remove free tracking agent and then observed using CLSM.

Immunofluorescence analysis was performed as previously described⁴⁸ (link) with minor modification. In brief, cells were seeded in glass-bottomed dish (#801002; NEST, Wuxi, China; 2.2 × 10⁵ cells/dish for J774A.1 and 1.5 × 10⁵ cells/dish for BMDMs) overnight, followed by priming with Pam3CSK4 (1 μg/mL) in Opti-MEM for 4 h. After pre-treatment with scutellarin (50 μmol/L) or MCC950 (2 μmol/L) for 1 h prior to transfection with 2.5 μg/mL LPS plus FuGENE-HD (2.5%, v/v) in Opti-MEM for 16 h, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, the cells were incubated with the anti-caspase-11 antibody (#ab180673) overnight, followed by incubating with CF568-conjugated goat-anti-rabbit IgG for 1 h. Subsequently, the cells were incubated with AlexaFluor488-conjugated anti-ASC antibody overnight. In some experiments, AlexaFluor488-conjugated anti-ASC antibody was used to reveal ASC expression. After being stained with Hoechst 33342 solution [5 μg/mL in phosphate-buffered saline (PBS)] to reveal the nuclei, the cells were observed under a Zeiss Axio Observer D1 microscope armed with a Zeiss LD Plan-Neofluar 100×/0.6 Korr M27 objective lens (Carl Zeiss).