Ds qimc

Monolayer wounding assays were used to evaluate migration of keratinocytes as described before [95] (link). Briefly, NHK cells were grown to confluence in ibidi μ-dishes containing culture inserts. Cells were incubated in Keratinocyte SFM® medium (with rhEGF, BPE and penicillin-streptomycin) containing 1 μg/mL TE or 10 ng/mL HGF. Live cell imaging migration was recorded in the PFS system on a Nikon Eclipse Ti microscope with a digital sight DS-QiMc (Nikon Instruments Inc., Tokyo, Japan), coupled to an ibidi-heating chamber. The migratory activity was measured by calculating the percentage of closed areas.

Organs were dissected, fixed in 10% phosphate-buffered formalin, and processed for paraffin sections. Four-micrometer sections were dewaxed and hydrated. Antigen retrieval was performed by microwaving tissue sections for 15 min in Target Retrieval Solution (DAKO). Tissue sections were incubated overnight with primary antibodies (Abs) in a humidified atmosphere at 4 °C. After washing, conjugated secondary Abs were added and then incubated for 35 min. The slides were washed and mounted with Fluoromount-G (SouthernBiotech). Primary and secondary Abs used are listed in Supplementary Table 1. Immunofluorescence imaging was performed in a fluorescence microscope (Nikon Eclipse Ni) with Plan Apo objective lenses. Images were acquired using a digital camera (DS-QiMc; Nikon) and Nis-Elements BR imaging software. After setting the acquisition conditions according the brightest sample, all the acquisition parameters were kept the same at all times, including the laser intensity, the exposure time, the gain of the photomultiplier, the offset of the histogram, and the image magnification. An average of three determinations were made for each mouse examined. The fluorescence intensity was measured by Image J/Fiji. Images were composed in Adobe Photoshop CS3.

Live phase contrast images from PC12 cells under the different conditions were acquired using a Nikon Eclipse Ti Inverted Microscope (Nikon; Düsseldorf, Germany) equipped with a Perfect Focus System (PFS) and a Digital cooled Sight Camera (DS-QiMc; Nikon, Germany) as described in (Weber et al., 2013 (link)). Briefly, PC12 cells were cultured in collagen coated 6-well plates (500,000 cells/well) and treated as described in “Cell culture and stimulation” and 150 images per well, every second day were recorded with the same spatial pattern. Cell differentiation is calculated by the ratio of the two described imaging features (Weber et al., 2013 (link)) convex hull (CH) to cell area (CA) for 150 images per well over 6 days (Weber et al., unpublished data).

Intracellular reactive oxygen species (ROS) were determined using the KITNAME (Sigma-Aldrich), as previously described [1 (link)]. Briefly, cells were seeded and incubated for 24 h, after which the culture medium was removed and replaced by fresh medium containing 1/10 IC₅₀, 1/5 IC₅₀ and IC₅₀ (0.08 µg. µL⁻¹), plus controls. Hydrogen peroxide (50 µM) was used as a positive control. Cells were harvested after 48 h, washed twice with PBS before resuspension in pre-warmed (37 °C) PBS containing 100 µM 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and incubation at 37 °C for 20 min, in the dark. Fluorescence intensity was measured on an Eclipse Ti inverted microscope equipped with a DS-QiMc camera and adapted for epifluorescence (all from Nikon), with fixed exposure time for all samples. The green fluorescent signal was normalized and analyzed using ImageJ [76 (link)].

Between 5000 and 6000 NHLF cells expressing α-SMA were plated in 12-well plates with silicone culture inserts (Ibidi, GmbH, Planegg, Germany) for 48 hours with either 5 μg/mL type IV collagen protein or 0.5 M acetic acid as a control. Inserts were removed, fresh treatment added, and cell migration monitored during 24 hours. Images of the different time points captured on a Nikon DS-QiMc camera using NIS-Elements BR 3.0. Quantification of cell invasion into the insert space was performed and compared using ImageJ software. Experiments were conducted three times (n = 3) in triplicate. Gap cell invasion was analyzed by two-tailed Student’s t test.

An inverted fluorescence microscope (Nikon Eclipse Ti, Japan) fitted with FITC 480/30 nm ex 535/45 nm em, Texas Red 560/40 nm ex 630/60 nm em, and DAPI 375/28 nm ex 460/60 nm em filters was used to visualise live and preserved cells in multi-well plates under 200, or 400× magnification and were imaged with a monochrome camera (Nikon DS-QiMc) using proprietary software (NIS Elements v4.60).
An upright fluorescence microscope (Nikon Eclipse Ni, Japan) fitted with a Cy5 620/60 nm ex 700/75 nm em filter and a monochrome camera (Nikon DS-Qi2) with proprietary software (Infinity Analyse v6.4.0) under 200, 400, or 1000× magnification was used for more detailed observation of cells and for obtaining cell size measurements.