Tcs sp5 confocal spectral microscope imaging system

Subcellular localization assays were performed as previously described (Chen et al., 2014 (link)). Briefly, plasmids containing 35S: CRKs-GFP or 35S: GFP were co-transfected with the plasma membrane marker pm-rkCD3-1007 (Nelson et al., 2007 (link)) into Arabidopsis mesophyll protoplasts by polyethylene glycol (Sigma)(Yoo et al., 2007 (link)). The samples were visualized 30 h after transfection using a TCS SP5 confocal spectral microscope imaging system (Leica).

The
LIVE/DEAD cell staining technique was performed using a commercial
kit (Thermo Fisher Sciences, USA) to assess the viability of cells
encapsulated within GelMA and GelMA@GF hydrogels under three different
media conditions: (i) complete M199 medium (M199), (ii) complete ADSC
medium (ADSC), and (iii) 50:50 (M199:ADSC) medium. The GelMA and GelMA@GF
hydrogels were prepared in a 24-well plate and placed in a humidified
incubator (37 °C, 5% CO₂) for 14 days. The media were
changed every 48–72 h. Following this period, the GelMA hydrogels
were washed twice with DPBS and then stained with a LIVE/DEAD solution
according to the manufacturer’s instructions. The samples were
then observed using a laser scanning confocal microscope (TCS SP5
Confocal Spectral Microscope Imaging System, Leica, Germany) (a slice
width of 4.99 um). The live cells were labeled with calcein-AM, which
emitted green fluorescence, while the dead cells were labeled with
BOBO-3 iodide and emitted red fluorescence. Image analysis software,
ImageJ, was utilized to quantify the cell viability of the encapsulated
cells within the culture media.

The surface morphologies of the electrospun nanofibres were characterized through field emission scanning electron microscopy (FE-SEM), using a JSM 6500F instrument operated at 15–20 kV. Fibre diameters were determined using Image-J image processing software. For each electrospun mat, at least 100 fibres were considered from three different images to calculate the average diameter. The roughness of the triple-blend fibrous mats was examined using an ultra-precision benchtop 3D optical profiler (UPBOP, Talysurf CCI LITE, Taylor Hobson). The distributions of the biomacromolecules on the fibre surfaces were investigated through laser scanning confocal microscopy (LSCM), using a Leica TCS SP5 confocal spectral microscope imaging system featuring a 100-mW Ar blue laser operated at 494 nm. A red fluorescent antibody (Cy5-Biotin, Click Chemistry Tools) was employed to determine the distribution of the biotinylated anti-EpCAM antibodies on the EBFMs. The streptavidin-modified EBFMs were placed in a Cy5-Biotin solution (5 μg/mL) at room temperature. The Cy5-Biotin-adhered EBFMs were subsequently observed through LSCM at 651 nm. The grafting density of anti-EpCAM could be estimated by the emission of Cy5-biotin at 651 nm of wavelength.

Neuro-2a cells were cultured in 4-well slide chamber (Merck Millipore, U.S.A.) at 10,000 cells per well. Cells were co-transfected with pCMV6-Bcr-myc and pEGFP-C3, pEGFP-Hap1A or pEGFP—Hap1B. After transfection, cells were washed with 1X PBS and fixed using 4% paraformaldehyde for 5 min at room temperature. Following washes, cells were permeablized and blocked with 0.1% Triton X-100, 3% BSA in PBS for 30–60 min at room temperature. Afterward the cells were incubated with primary mouse anti-Myc antibody overnight. The cells were then washed and incubated with a fluorophore-conjugated secondary antibody at 1:5000 for 2 hours at room temperature. After washes, the cells were incubated with 300 ng/ml DAPI in 1X PBS for 10 min at room temperature. With sufficient washes, the chamber was disassembled, and the slide was air-dried and mounted. The slide was covered and sealed, and then visualized using TCS SP5 Confocal Spectral Microscope Imaging System (Leica, Germany) at Taipei Medical University Instrument Center. Images of co-localized area were generated using Image J 1.48v (Wayne Rasband, NIH, U.S.A), and image densities were quantified using Un-Scan-It 6.1 (Silk Scientific Crop., U.S.A.).

RAW 264.7 cells (5 × 10⁴ cells per well) were cultured on cover slips in 6-well plates and treated with 0.1% DMSO or 20 μM rutaecarpine with or without LPS stimulation for 30 min. The cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After incubation, the cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA for 30 min. The cells were incubated with primary antibodies overnight at 4 °C, subsequently washed 3 times with PBS, and incubated with secondary antibodies for 1 h at room temperature. The samples were stained with 4,6-diamidino-2-phenylindole (DAPI, 30 μM) and mounted using a mounting buffer (Vector Laboratories) on a glass slice. The samples were detected under a Leica TCS SP5 confocal spectral microscope imaging system using an argonor krypton laser (Mannheim, Germany).

For BiFC assays, plasmids of Pro35S: FLS2-YFP^N (modified pEarleyGate201, Huang et al., 2014 (link)) and Pro35S: BAK1-YFP^C (modified pEarleyGate202, Huang et al., 2014 (link)), Pro35S: FLS2-YFP^N and Pro35S: CRKs-YFP^C, Pro35S: RCI2B-YFP^N and Pro35S: RCI2B-YFP^c, or Pro35S: RCI2B-YFP^N and Pro35S: CRKs-YFP^C were transformed into Arabidopsis protoplasts by polyethylene glycol (Sigma) for transient expression (Yoo et al., 2007 (link)). The samples were visualized after treatment with 100 nM flg22 using a TCS SP5 confocal spectral microscope imaging system (Leica).

For BiFC assays, full length coding regions of BRM and BP were subcloned into YN vector pUC-pSPYNE and the YC vector pUC-pSPYCE, respectively [27 (link)]. Then fused YN and YC constructs were transformed into tobacco cells by polyethylene glycol for transient expression [56 (link)]. Transfected protoplast cells were imaged using a TCS SP5 confocal spectral microscope imaging system (Leica).