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www.nature.com/scientificreports/ stained with 4′,6-diamidin-2-phenylindol (DAPI) subsequently for 4 hours at 4 °C. The amount of migrated cells away from beads to the surrounding fibrin matrix was quantified by counting DAPI-positive cells in a determined region around the beads. HUVEC/ASC co-cultures in 3D fibrin matrices. 3D co-cultures of GFP-HUVEC and human ASC in fibrin gels were prepared as described previously7, 18, 19. In brief, both cell types were enzymatically detached with trypsin, counted, adjusted to a volume of 95 μl and mixed with 5 μl of 100 mg/ml fibrinogen (Baxter, Vienna, Austria) to a total volume of 100 μl. GFP-HUVEC and ASC were either used in a 1:1 ratio (105 cells per cell type in each gel) or in a 1:0.01 ratio (105 HUVEC seeded with 103 ASC). A 1:0.01 seeding ratio was chosen to be able to observe even small changes of vasculogenic events whereas a 1:1 seeding ratio usually leads to a mature capillary network after one week even without stimulation. Therefore, it was possible to detect and quantify single-cell specific changes in morphology and proliferation (1:0.01 co-cultures) as well as influences of LED light therapy on the formation of mature networks. Quantification of fluorescent cells and vascular networks. In order to quantify the vascular structure for- mation in light-stimulated GFP-HUVEC/ASC fibrin matrices, images were taken on day 4 and 7 (4 images per clot) on a Leica DMI6000B epifluorescence microscope (Leica, Solms, Germany) in a resolution of 1392 × 1040 pixels and stored in a TIF format. These timepoints were chosen to evaluate early vasculogenic events (day 4) but also maturation of the capillary network formed (day 7). For quantifying cell morphology changes in co-culture ratios of 1:0.01 after 4 and 7 days of incubation, Cellprofiler software version 2.11 was used. Initially a color to gray conversion was performed to obtain grayscale images. Subsequently Cellprofiler’s object detection routine was used to detect the individual cells. No further processing of the images was required. The build in “global background” threshold routine with a threshold correction factor of 2 was used to obtain accurate detection of the cells. The total number of cells, the fraction of area covered with cells and the form factor of each cell was measured. The form factor is defined as 4 * π * area/perimeter2 (it equals 1 for a perfectly circular object) and represents cell elongation. The higher the form factor, the less elongated cells are. The obtained data was stored in comma separated values format and statistically analyzed. NO measurement. Supernatants of 1:0.01 HUVEC/ASC co-cultures were collected after 4 days of incubation and subjected to NO measurement. To assess the synthesis of nitric oxide (NO) after illumination, 2.4 × 104 HUVECS were seeded in 24-well plates and grown to 80% confluence. The cells were illuminated and supernatant/medium samples were taken immediately and 4 days post-illumination. Nitric oxide was quantified with Sievers 280i-NO Analyzer (General Electrics) as previously described (Weidinger et al., Antioxid Redox Signal. 2015 Mar 1;22(7):572-86). Briefly, samples were injected into a glass vessel, containing vanadium chloride, a redox active reagent, which converts NO species into NO; this in turn reacts with ozone (O3) and gives a chemiluminescence signal. Protein expression. To determine protein expression changes mediated by LLLT in HUVEC:ASC co-cultures (1:0.01 ratio), supernatants of co-cultures were taken after 4 days of incubation and measured with an angiogenesis protein array kit (RnD systems, Minneapolis, USA) by investigating 55 different angiogenesis-related cytokines. Proteins which were ≥1.5 fold upregulated in light stimulated groups compared to control were con- sidered. The protein blots were developed with an exposure time of 5 min. ROS formation. For measurement of hydrogen peroxide formation, HUVEC were seeded in four wells of four 24-well plates at a density of 2.5 * 104 cells/well. 24 hours later, at 70–80% confluence, cells were used for treatment. The medium was aspirated and replaced by 300 μL Krebs-HEPES buffer per well after two wash- ing steps with 750 μL PBS/well. For inhibition of NADPH oxidase 3 μL of Diphenyleneiodonium (DPI – Sigma Aldrich, Germany, D2926), were added to two wells per plate to yield a final concentration of 1 μM. Each plate was then randomly assigned to one group and irradiated accordingly for 10 minutes at a distance of 2 cm, while the control plate was wrapped in tin foil. Samples were taken and measured 5 min, 15 min and 30 min after treatment. For measurement 50 μL of the supernatant were transferred into two corresponding wells of a black, opaque 96-well plate (Greiner Bio-One GmbH, Austria) and 50 μL of Amplex® Red (Thermo Fisher Scientific Inc., Waltham, Massachusetts, A12222) horseradish peroxidase (HRP – Sigma Aldrich, Germany, P8250) solution was added. The color reaction was analyzed using a POLARstar® Omega plate reader (BMG LABTECH GmbH, Germany) by measuring the fluorescent emission at 590 nm (excitation wavelength at 544 nm). Statistics. Statistical differences were evaluated by using one-way ANOVA tests with Dunnett post testing to assess significances with prior evaluating the presence of Gaussian distributions. All data sets are presented as mean ± stand- ard deviation. P-values less than 0.05 were considered as significant. All statistical analyses were performed with GraphPad Prism 4.0 software (GraphPad, San Diego, USA). All experiments were performed 6 times with HUVEC from two different single donors and one pooled donor HUVEC sample to reduce variation in combination with 3 diverging ASC donors for co-culture experiments. N = 6 for all experiments except the evaluation of protein expression after 4 days which was performed with pooled samples from three donors in duplicate. References 1. Rochkind, S. et al. Efficacy of 780-nm Laser Phototherapy on Peripheral Nerve Regeneration after Neurotube Reconstruction Procedure (Double-Blind Randomized Study). Photomedicine and Laser Surgery 25, 137–143 (2007). 2. Aimbire, F. et al. Low-Level Laser Therapy Induces Dose-Dependent Reduction of TNFα Levels in Acute Inflammation. Photomedicine and Laser Surgery 24, 33–37 (2006). 3. Silva Júnior, A. N. et al. Computerized Morphometric Assessment of the Effect of Low-Level Laser Therapy on Bone Repair: An Experimental Animal Study. Journal of Clinical Laser Medicine & Surgery 20, 83–87 (2002). 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