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www.nature.com/scientificreports/ components coming into play. In our previous reports in in vivo models, in which blue light was effective, wound healing has been disturbed by ischemia. In this setting oxygen-independent nitrite synthases are active to bio- activate nitrite to NO and light can influence the activity of these enzymes33. Blue light is also most efficient to release NO which is bound to mitochondrial and other heme proteins5, 13. NO has been shown to play a key role in wound healing. Also light-induced NO-dependent vasodilation takes place in vivo34. All these mechanisms do not occur in in vitro systems. Therefore it is not remarkable that there are differences between in vitro and in vivo. However, in vitro studies are valuable and necessary to investigate the role and effects of selected cell types. Thus, although insight into possible mechanisms was gained in the last few years by performing in vitro investigations35, the influence of different wavelengths may still hold great potential. Methods Cells. All experimental protocols were approved by the Ethics Committee of the Land Upper Austria. The informed consent was obtained from all subjects. The methods were carried out in accordance with the approved guidelines. HUVEC were either purchased from Lonza (C2519-A or C2856; Lonza, Basel, Switzerland) or isolated from fresh umbilical veins as described before36. Green fluorescent protein (GFP) expressing HUVEC were purchased from Olaf pharmaceuticals (GFP; Olaf pharmaceuticals, Worcester, USA). The collection of human adipose tis- sue was approved by the local ethical board with patient’s consent. Subcutaneous adipose tissue was obtained during routine outpatient liposuction procedures under local tumescence anaesthesia. Human ASC were isolated from liposuction material as described before37, 38. HUVEC, GFP-HUVEC and ASC were cultured in endothe- lial growth medium (EGM-2; Lonza) supplemented with 5% fetal calf serum (FCS; GE Healthcare, Chalfont St Giles, UK) at 37 °C with 5% CO2. Endothelial cells were maintained in cell culture flasks (TPP, Trasadingen, Switzerland) coated with 2 μg/ml bovine fibronectin (Sigma-Aldrich, St. Louis, USA) whereas ASC were cultured on uncoated plastic surfaces (TPP). For experiments cells were seeded in 24-well plates. LLLT stimulation. LED lamps for light therapy were provided by Repuls Lichtmedizintechnik GmbH, Austria. Cells were treated with pulsed LED light of either 475 nm (blue), 516 nm (green), 635 nm (red) or remained unstimulated (control). All LED devices had a peak irradiance intensity of 80 mW/cm2 which was measured with a USB 2000 spectrometer (Ocean Optics, FL, USA). Given the pulse rate of 50% and a repetition frequency of 2.5 Hz an average irradiance intensity of 40 mW/cm2 was reached. The daily dose provided was 24 J/ cm2. Illumination was performed at room temperature for 10 min at a distance of 2 cm from the cells. For 2D experiments, cells were stimulated on day 0 only whereas for 3D experiments stimulation was performed every 24 hours (Fig. 1). Proliferation and MTT assays. To assess the effects of light treatment on HUVEC proliferation, 104 HUVEC were seeded to 24-well plates for each condition per time point and grown until 20% confluence. Cells were stimulated by LLLT on day 0 and further cultivated for up to 72 hours. Every day duplicates of cells were enzymatically detached with trypsin (Sigma-Aldrich) and counted with a Neubauer counting chamber (VWR, Darmstadt, Germany). To assess the effects of light treatment on HUVEC cell metabolic activity assay, HUVEC were as described above. However, instead of detaching, every day duplicates of cells subjected to MTT assays. Briefly, the superna- tants were removed and cells were washed three times with PBS. MTT reagent (Sigma) was dissolved in 1× PBS (5 g/L). Before adding to the cells, this stock solution was diluted with the respective growth medium to 3.25 g/L and the plate was incubated for 1 hour at 37 °C. The supernatant was removed and formazan crystals were dis- solved using dimethyl sulfoxide (Sigma). The plate was incubated on a shaker in the dark at room temperature for 20 min and absorbance was measured at 540 nm with 650 nm as reference. 2D migration- Scratch assay. Scratch assays were performed as described previously7. Briefly, 104 HUVEC were seeded to wells of a 24-well plate and grown until 100% confluence. To investigate migration in a 2D mon- olayer, an artificial wound was created by scraping the cells off the well with a 1000 μl pipet tip (Greiner bio one, Kremsmünster, Austria) within a certain area. Scraped cells were removed by washing the monolayer twice with 1x phosphate buffer saline (PBS). 1 ml of EGM-2 medium was added to each well and LLLT was performed immediately. Images of the scratched wounds were taken directly after stimulation (0 h) and after a 6 hours incu- bation time at 37 °C. Three images per scratch were taken to cover the whole scratched area of each well. In order to quantify the cell migration into the artificial wound, the cell-free area was quantified with ImageJ using the area measurement tool. 3D migration- bead assay. To analyze the effects of LLLT on 3D migration of HUVEC, cell-coated Cytodex 3 beads (GE Healthcare) were incorporated into 3D fibrin matrices and the migration of cells from the beads into the fibrin matrix was quantified. Therefore, cells at a seeding concentration of approximately 100 cells per bead (2 × 105 beads/ml) were incubated with beads in EGM-2 for 4 hours at 37 °C with occasional shaking every 20 min to ensure an even cell coating of the beads. The beads were then incubated overnight. The next day, 5 μl bead suspension (approximately 100 beads) was added to 5 μl of fibrinogen (100 mg/ml; Baxter, Vienna, Austria) and 90 μl EGM-2. The fibrinogen solution containing the cells was then mixed with 100 μl thrombin (0.4 U/ml, Baxter, Vienna, Austria) resulting in fibrin gels with a final fibrin concentration of 2.5 mg/ml and a total volume of 200 μl. These matrices were prepared on round coverglasses with 15 mm diameter (VWR, Radnor, USA) in 24 well plates. After polymerization at 37 °C for 20 min EGM-2 was added to the wells. Gels on the coverglasses were stimulated 2 hours after polymerization of the fibrin gel and subsequently every 24 hours until measurements and quantifications were conducted. The fibrin gels were stimulated every day for 4 consecutive days. The gels were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 6 hours at 4 °C while shaking and cell nuclei were SCientifiC REpORTS | 7: 10700 | DOI:10.1038/s41598-017-11061-y 8PDF Image | impact of wavelengths of LED light-therapy on endothelial cells
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