Tcs sp8 x aobs

The fluorescence of the cells was analyzed by flow cytometry after treating with PNPs and DPNPs containing 1 μM AN2728 for 8 h. The isotype control of DPNPs was nanocarriers decorated with lymphocyte antigen 6 complex locus G6D (Ly6G) antibody. The conjugation method for anti-Ly6G antibody was the same as that described above for anti-Dsg3 antibody. Fluorescence from a gated population of HaCaT cells labeled with rhodamine 800 was acquired on channel FL3 (> 650 nm) with excitation by a 488 nm solid-state laser. Data were collected from at least 10,000 cells at a flow rate of 35 μL/min. A logarithmic scale was used to measure both background and cell fluorescence. Cell internalization of rhodamine 800-labeled nanohybrids was also assessed by confocal fluorescence microscopy (Leica TCS SP8 X AOBS). Cells were labelled with 4′,6-diamidino-2-phenylindole (DAPI) and LysoTracker Green DND-26 according to the manufacturer's instructions (Invitrogen). Images were pseudocolored blue for DAPI, green for LysoTracker, and red for rhodamine 800. The fluorescence intensity of rhodamine 800 was quantified from the captured images using ImageJ software.

To evaluate the nanoparticle uptake by 3T3-L1 cells, free CLA or CLA-loaded NLCs were incubated with the cells (1 × 10⁵ cells/well) for 12, 24, and 48 h. The harvested cells were centrifugated at 500 ×g for 10 min, and then the cell pellet was lysed with lysis buffer (0.5 ml/well). The intracellular CLA content was quantified by LC/MS. The nanoparticles were also labeled with fluorescence dye (10 µg/ml rhodamine 800) to detect nanoparticle internalization in cells by flow cytometry and confocal microscopy. After incubation of the cells with the dye-loaded nanoparticles for 24 h, fluorescence from a gated population of 3T3-L1 cells (10,000 cells) was acquired on channel FL3, with excitation at a wavelength of 488 nm. The cell uptake of nanoparticles was also monitored by confocal microscopy (TCS SP8 X AOBS, Leica). The adipocytes were stained with 4’,6-diamidino-2-phenylindole (DAPI) and LysoTracker (Invitrogen) to observe the nuclei and lysosomes, respectively.

HaCaT cells were grown on coverslips at a density of 1 × 10⁵ cells/mL, treated with the indicated treatments, then fixed in 4% paraformaldehyde for 30 min. Cells were permeabilized for 15 min at room temperature with PBS containing 0.2% Triton X-100 and then blocked with 2% BSA for 1 h. The coverslips were then incubated overnight with NF-κB p65 primary antibody (1:200 dilution, GeneTex) at 4 °C, then treated with goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488 (1:1000 dilution, Invitrogen) for 1 h. The coverslips were mounted onto glass slides using Slowfade^® Gold Antifade Mountant with DAPI and examined by confocal laser scanning microscopy (Leica TCS SP8 X AOBS).