710 nlo

The cell viability was measured by WST-1 assay according to the manufacturer's protocol (Roche Applied Sciences). 5 × 10³ cells were plated on 96-well plates and after overnight incubation treated with the indicated drugs for 72 hours. Cell viability defined as the absorbance in the treatment group compared to the control group was expressed as percent mean change ± SD (n = 4). Apoptosis was assessed using differential staining with acridine orange (green) (2 μg/ml; Sigma-Aldrich) incorporated by all cells and ethidium bromide (red) (2 μg/ml; Promega) incorporated only by apoptotic cells with compromised cell membrane. Quantification of apoptotic cells co-stained with acridine orange and ethidium bromide was performed on the images taken with confocal microscope Zeiss NLO710 as described [26 ]. Four replicate experiments were performed. Cells were counted in 5 independent random view fields.

AlexaFluor647-labelled NP were conjugated to 3-Rhod, purified and concentrated, and then 30 μL of the resulting solution, containing 1.8 mg of NPs, was injected i.d. in both forearms of three 6 wk old female C57BL6/J mice. After 24 h the mice were sacrificed and the axillary and brachial LN excised, cleaned under stereoscopic observation, and plated on a glass-bottom 96-well plate for confocal imaging (Zeiss NLO 710). Using a 10x objective lens, 16-bit fluorescence imaging was taken for AlexaFluor647 and rhodamine. Z-stacks approximately 6.4 μm in depth were taken through the depth of the LN (approx. 160 μm). In order to acquire the entire LN, convex hull tiling was performed and the resulting images were stitched together.

The 250,000 cells were plated in glass coverslips in 6-well plates and allowed to attach for 24h at 37°C (5% CO₂). The cells were incubated in 1:5000 concentration of Mitotracker Deep Red (Invitrogen) for 15 minutes, washed with prewarmed media, then fixed and permeabilized in 100% methanol for 5 minutes at 4°C. Next, the cells were blocked with 5% BSA in PBS-TT (0.5% Tween and 0.1% Triton) for 1h and immunostained for mGPDH using monoclonal antibody anti-mGPDH (Abcam, Cambridge, MA) for 2h then secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 569 for 1h (Life Technologies, Thermo Fisher Scientific). DNA was stained with DAPI (Vector Laboratories, Burlingame, CA). Fluorescence images were detected by confocal microscope NLO 710 Zeiss, and collected using Carl Zeiss Zen Software (Zeiss, Germany).

Upon completion of biaxial mechanical testing, the tissue samples were imaged using the SHG technique at the unloaded state. We utilized a Zeiss 710 NLO inverted confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA), equipped with a mode-locked Ti:Sapphire Chameleon Ultra laser (Coherent Inc., Santa Clara, CA), a non-descanned detector (NDD), and a Plan-Apochromat 40× oil immersion objective. The laser was set to 800 nm and emission was filtered from 380–430 nm. Samples were kept hydrated with saline solution during imaging to prevent drying artifacts and covered with #1.5 coverslips. Samples were imaged inside the area delimited by the graphite markers, and 2D image slices were collected in the thickness direction from the smooth side of each sample. A 2D slice has 512×512 pixels to 1024×1024 pixels, and for each sample the number of slices was varied to cover the thickness. In total, we obtained 3D SHG images (size from 512×512×N to 1024×1024×N) of 48 tissue samples from different animal subjects, and the corresponding mechanical testing data. Representative SHG images of a GLBP sample are shown in Figure 2, with a total of 18 slices (N=18) through the thickness. It can be seen that geometry variation (e.g. fiber waviness) in each imaging plane is much larger than that in the thickness direction as shown in Figure 2.