Dm ire2 confocal microscope

Distribution of Rf-LPN in biofilms was visualized by confocal laser-scanning microscopy (CLSM). The bacteria suspension was incubated in 96-well plate overnight. Rf-LPN labeled with fluorescent DPPE-lissamine rhodamine B were added to the wells. We limited the amount of fluorescent-phospholipid used to be 0.25 %w/w by total weight of the nanoparticles. At this quantity we observed no significant differences in particle size, drug loading and release behaviors, cytotoxicity and rifampicin uptake performance (data not shown). After 12 h, each well was washed with PBS three times and stained with 10 μg/ml wheat germ agglutinin-Alexa Fluor 633 for 15 min to indicate the biofilm structure. The samples were fixed with 4% formaldehyde before scanned with Operetta CLS High-Content Analysis System (PerkinElmer, Walham, MA).
To visualize distribution of Rf-LPN in osteoblasts, the cells grown on poly-lysine treated coverslips were treated with 50 μl of 10 mg/ml Rf-LPNs labeled with 0.25 %w/w DPPE-lissamine rhodamine B for 6 h at 37 °C. The osteoblast nuclei and cell membrane were stained with DAPI and wheat germ agglutinin Alexa Fluor 633 Conjugate, respectively. The cells were then washed with PBS, fixed with 4% formaldehyde for 10 min, mounted on slides with Fluoromount and visualized with a Leica DM IRE2 confocal microscope with a TCS SL system.

To visualize the distribution of live and dead cells in the scaffolds, the cells were stained with a 4µM Ethidium homodimer-1 and 2µM calcein AM solution. The cells were incubated for 30–45 minutes at room temperature and then imaged on an inverted Leica DMIRE2 confocal microscope.

For ICC, cells were plated on 50 μg/mL PDL-coated 12-mm glass coverslips. Cells were washed with PBS and incubated in 4% paraformaldehyde for 30 min at RT. After fixing, the cells were washed with PBS and incubated in blocking agent (2% BSA, 0.05% Tween-20, and 0.5% Triton X-100 in PBS) for 45 min. Cells were then incubated with antibodies against human αSyn (Syn 211; Santa Cruz, 1:500) and GFP (Abcam 1:2000) overnight at 4°C or the cytoskeleton marker Phalloidin (Alexa Fluor 647 phalloidin, Invitrogen) for 30 min at RT. After primary incubation, the cells were washed and incubated in the dark for 90 min with Alexa-488 and -555 dye-conjugated secondary antibodies (Invitrogen, 1:1000). Hoechst 44432 was used as a nuclear stain and the coverslips were then mounted on glass slides and viewed with 63× and 43× oil objectives using a Leica DMIRE2 confocal microscope.