Tcs sp5ii mp smd flim

Six-week-old BALB/c mice were anesthetized by subcutaneously injecting a mixture of 40 μL of ketamine and 10 μL of rompun in 200 μL of PBS.^{26 (link)} After 20 min, the mice were anesthetized and the hair of the back portion to be inserted with a MN was removed with an electrical clipper and hair removal cream. FITC-labeled NPs were synthesized by the reaction of FITC and F127–NH₂. Briefly, F127–NH₂ was dissolved in THF, then 2 molar excess of FITC was added. The mixture was stirred for 2 h and purified by dialysis and lyophilization. The MN loaded with FITC-labeled NPs was inserted into the back of mice. After 1 and 24 h, the MNs were removed, respectively, the mice were sacrificed, and the back skin tissues were dissected. The back skin tissues were observed by two-photon fluorescence microscopy (TCS SP5II MP SMD FLIM, Leica, Deerfield, IL). The images were obtained by skin tissue tomography (Z-stacks), 512 × 512 pixels, and reconstructed with LAS AF Lite 2.6.1 (Leica) and ImageJ program.

The 5 × 10⁵ murine macrophage cells (RAW 264.7) were incubated in a 6-well microplate for 24 h. After adhesion onto a microplate, the culture medium was replaced with a serum-free medium. FITC labeled F127–R837@M NPs (10 μg) were dropped into each well for 24 h. To label the nucleus of RAW 264.7, aqueous mounting medium was dropped for 4 h. The cells were washed with PBS and fixed in a 10% formaldehyde solution. The cells were observed by confocal microscopy (TCS SP5II MP SMD FLIM, Leica, Deerfield, IL). Further cellular uptake test was performed by measuring the amount of R837 in the cell media with HPLC. After 1 and 24 h, each cell media was aspirated and analyzed. Cell viability test was performed by MTT assay at various concentration of NPs (1, 5, 10 and 20 μg mL⁻¹) using the same procedure with the cellular uptake experiment.