Nunc lab tek chamber

B16-F10 cells at a density of 2 × 10⁴ cells/well were seeded in eight-well glass Nunc Lab-Tek^® chamber (ThermoFisher Scientific, Waltham, MA, USA) and treated FRK with or without LEO or REO for indicated time points. After treatment, culture media was removed and the cells were fixed in 2% paraformaldehyde for 15 min, cells were permeabilized with 0.1% Triton X-100 for 10 min, washed and blocked with 10% FBS in PBS and then incubated for 2 h with the anti-phospho-CREB or anti-MITF primary antibodies in 1.5% FBS. The cells were then incubated with the fluorescein isothiocyanate (FITC)-conjugated secondary antibody for another 1 h in 6% bovine serum albumin (BSA). Next, the cells were stained with 1 μg/mL DAPI for 5 min, washed with PBS and visualized using a laser scanning confocal microscope (Leika Microsystems, Buffalo Grove, IL, USA) at a 20 × magnification.

In brief, 400 μL of approximately 10⁷ CFU/mL S. aureus or S. enteritidis was added to each well of the Nunc™Lab-Tek™ chamber slides (ThermoFisher Scientific, Kamstrupvej, Denmark). The broth was changed every 24 h to keep the bacteria active. The biofilms in the chambers were treated according to the method in Section 2.3. The biofilms were dyed and observed according to previous reports [20 (link),21 (link)].

Supported lipid bilayers (SLBs) were prepared as previously described²³ . First, 1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl) iminodiacetic acid succinyl] (nickel salt) (DGS-NTA(Ni)) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine were dissolved in chloroform and mixed in a 1:50 molar ratio. The mixture was then dried under vacuum overnight and resuspended in degassed PBS. Sonication was performed under nitrogen until the suspension became clear. Nonunilamellar vesicles were pelleted through ultracentrifugation, and the clear supernatant was subjected to further centrifugation. The second supernatant was filtered and stored under nitrogen. Glass slides were cleaned for 15 min using plasma (Zepto, Diener Electronic). Cleaned slides were attached to the bottom of an 12-well Nunc Lab-Tek chamber (Thermo Fisher Scientific) with Picodent twinsil extrahart (Picodent) until the glue had solidified. The lipid vesicle suspension was diluted 1:20 with PBS and filtered, and 100 μL of the diluted suspension were added to each well to form a continuous SLB. Excess vesicles were removed by washing the chambers with PBS. H12-tagged proteins were added to the SLBs and incubated for 60 min in the dark at room temperature. Finally, the chambers were rinsed with PBS to remove unbound protein.

Different types of UPy-aggregates (Table S1, ESI† 10 μM) containing one percent of UPy–Cy5 as a reporter were prepared in DMEM and RPMI medium accordingly. THP-1 and HK-2 cells were seeded in an 8-well Thermo Fisher Scientific™ Nunc™ Lab-Tek™ Chamber with #1 borosilicate glass (n = 4). After attachment, the cells were washed three times with PBS and 0.4 mL of UPy-aggregates suspension was added into each well. For the experiments in the presence of FBS, the UPy-aggregates were incubated with cells for 5, 30 and 120 min. For the studies in the absence of FBS, the UPy-aggregates were incubated with cells for 120 min. At each time point, the UPy-aggregates were washed away with PBS, followed by sequential staining with Hoechst 33342 and CellMask™ Green plasma membrane stain for nuclei and membranes, respectively. After staining, the cells were washed three times with PBS and Invitrogen™ Live Cell Imaging Solution was added into each well for live imaging. The live imaging was performed under a Leica SP5 CLSM at 37 °C.

Schwann cells transfected with siRNA against MOGS or a negative control were cultured in a Nunc™ Lab-Tek™ chamber (ThermoFisher Scientific, Waltham, MA, USA). Time-lapse images were taken with an Olympus IX81 microscope (Olympus, Tokyo, Japan). Images were taken every 5 min during the 12 h of observation. Xcellence Sequence (Olympus) and ImageJ (National Institutes of Health, Bethesda, MD, USA) were used to determine cell migration distance and velocity, respectively.

CLSM live imaging with the mitochondria staining was performed to explore the location of the UPy-aggregates after incubation with cells. Cationic UPy-aggregates (Fig. 1(b) and Table S1, ESI† 10 μM) containing one percent of UPy–Cy5 as a reporter were prepared in DMEM medium in the presence or absence FBS. THP-1 and HK-2 cells were seeded in an 8-well Thermo Fisher Scientific™ Nunc™ Lab-Tek™ Chamber with #1 borosilicate glass bottom (n = 4). After attachment of the cells, 20 and 8 μL of CellLight® Mitochondria-GFP BacMam 2.0 Reagent were added into each well for THP-1 derived macrophages and HK-2 cells, respectively, to induce the mitochondria GFP secretion. The cells were then washed three times with PBS after overnight culture. The cationic UPy-aggregates (0.4 mL) in media with or without FBS were added and the cells were incubated for 120 min. After that, the cells were gently washed three times with PBS and stained by Hoechst 33324. Location of the cationic UPy-aggregates was then observed under CLSM live imaging at 37 °C.