Nunc lab tek 2

Hela cells were plated on chambered coverglass (Thermo Scientific Nunc Lab-Tek II) and cultured overnight in the maintenance medium in humidified atmosphere at 37 °C with 5% CO₂. Cells were incubated with 20 μg mL⁻¹ of fluo-CNOs for 2, 5, 12, 24 and 48 h. As a control, the cells were left untreated (data not shown). After incubation, the cells were rinsed three times with phosphate buffered saline (PBS) (0.1 M, pH 7.4), fixed in a combination of paraformaldehyde (3%) and PBS and incubated with a solution of Hoechst 33342 (5 μg mL⁻¹) (Sigma) for 20 min. Finally, the cells were rinsed three times and filled with PBS. Confocal fluorescence imaging was then carried out with a Nikon A1R laser scanning microscope and a plan apo 20× DIC M and a plan apo VC 60× oil DIC N2 objective. In order to switch the fluorescence on and off, the cells were incubated respectively with an acidic PBS solution (pH 4.5) and a basic PBS solution (pH 8.5) for 1 h before imaging.

Rat tail collagen type-1 (Corning, Inc. NY) was purchased from the manufacturer as a concentrated stock solution. In order to study collagen in macromolecular form, the stock solution was diluted to a final collagen concentration of 3 mg/mL in 20 mM acetic acid. The resulting molecular solution of collagen remained unpolymerized when kept at room temperature for the duration of experiments. In addition, hydrogels exhibiting fibril and matrix-level structures of collagen were prepared according to manufacturer’s protocol. Appropriate amounts of 10X Dulbecco's Phosphate-Buffered Saline (10X DPBS, Life Technologies), 1.0 N NaOH, cell culture grade distilled water, and collagen stock solution were mixed together in a centrifuge tube on ice in the given order, resulting in a solution with neutral pH and isotonic ionic strength. This neutralized collagen solution had a final collagen concentration of either 3 or 6 mg/mL. Prepared solution was then dispensed into a chamber slide (Nunc Lab-Tek II, Thermo Scientific) and allowed to polymerize for 1 hour at 37°C in a CO₂ incubator. Afterwards, isotonic saline solution (1X DPBS) was added on top of the hydrogel to prevent dehydration and incubation was continued until the experiments next day.

GL261 cells were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II, Thermo Fisher Scientific, USA) at a density of 5 × 10⁴ cells/well. After 24 h culture, the cells were treated with the varying concentrations of fluorescent coumarin-labelled polymersomes. At the exposure time t = 30 min, 2 h or 4 h, the cells were pre-stained with LysoTracker Red (final concentration, 1 μM) for 30 min at 37 °C. Subsequently the cells kept in the PBS (10% FBS, v/v) were immediately observed using confocal laser scanning microscopy (FV3000, Olympus, JPN). The images were analyzed with software Image J (NIH, USA).

SH-SY5Y neuroblastoma cells (ATCC; CRL-2266) were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 media (1:1) (Thermo Fisher) containing 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin in an incubator (37 ℃, 5% CO₂) as previously described (53 ). When the cells reached 70 to 80% confluency, they were transfected with plasmids for ADRB1 or PSD-95 using polyethylenimine (Polysciences). After 24 h, cells were harvested for further analysis. For SH-SY5Y differentiation, cells were cultured in a 4-well chamber slide (Nunc™ Lab-Tek™ II, Thermo Scientific) with DMEM/F-12 media. The next day, cells were treated with 5 µM RA to induce differentiation for 5 d, and the medium was changed every other day. At day 6, the cells were transfected with plasmids. After 24 and 48 h, the cells were fixed for immunofluorescent staining.