F viewii firewire camera

After sterilization of PLA-based films, stem cell suspensions of 1.5 × 10³ of hBM-MSCs and hASCs were seeded on each film surface, and then 500 μL of culture medium was gradually added. As a CTR experiments were performed seeding stem cells on glass coverslip (GC). The morphology of stem cells was analysed by immunostaining of the cytoskeleton at different time points (3D, 7D and 14D). Briefly, the stem cells on different films were rinsed twice with PBS, fixed in 4% paraformaldehyde for 20 min, rinsing with PBS, permeabilized (PBS + 3% FBS + 0.5% Triton X-100) and finally blocked (PBS + 3% FBS + 0.05% Triton X-100) for 1 h at r.t. To achieve F-actin fibres, samples were incubated with phalloidin (Alexa-fluor-488 phalloidin, Invitrogen, Grand Island, NY, USA) for 20 min at r.t. and then after washing with PBS, samples were mounted and nuclei were counterstained with Vectashield^® with DAPI. Image acquisition was performed by using a fluorescence microscope (Eclipse-TE2000-S, Nikon, Tokyo, Japan) equipped with the F-ViewII FireWire camera (cell Soft Imaging System, Olympus, Germany, version 2.5, Accessed in 2006). PLA films without cells were also evaluated to analyse their interference.

Immunostaining was performed as previously described [17 (link),21 (link),42 (link),43 (link),75 (link)]. Briefly, stem cells on PBCE*, PBCE, BGD10 and BDG30 squares, and on TCP and GC were rinsed twice with PBS, fixed in 4% paraformaldehyde for 20 min, washed twice with PBS, and permeabilized and blocked (PBS + 3% FBS, 0.3% Triton X-100) for 1 h at RT. Samples were incubated with phalloidin (Alexa-fluor-488 phalloidin, Invitrogen) for 20 min at RT, or overnight at 4 °C with the primary human antibodies: anti-MAP2 (Abnova, Taipei, Taiwan), anti-GFAP, anti-NG2 (Millipore, Billerica, MA, USA), anti-TJU1, anti-nestin, and anti-βTubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In the latter cases, after 3 washing with PBS, the staining with Alexa-Fluor 488-nm or Alexa-Fluor 594-nm conjugated secondary antibodies (Invitrogen) for 1 h at room temperature was performed. After being washed with PBS, samples were mounted and nuclei were counterstained with Vectashield with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Images were acquired using fluorescence microscopy (Eclipse-TE2000-S, Nikon, Tokyo, Japan) equipped with the F-ViewII FireWire camera (Soft Imaging System, Olympus, Münster, Germany). Images were elaborated as described below. Interference of PBCE*, PBCE, BDG10 and BDG30 squares without cells to a fluorescence microscope (Eclipse-TE2000-S) was evaluated.

Fluorescent stained images were used to calculate the parameters and morphometric descriptors by Fiji software (Fiji Life-Line version, 30 May 2017), as previously described [14 (link)]. Images were acquired using a fluorescence microscope (Eclipse-TE2000-S, Nikon) equipped with the F-ViewII FireWire camera (Soft Imaging System, Olympus).
For the morphometric descriptors analysis, eight different areas were photographed (20X magnification, x20 Plan Fluo NA 0.5) and a total of 100 nuclei and cells were analyzed, for each polymer film. The area, the perimeter, the major and minor axis of nuclei and cells were measured to quantify the variation of the nuclear shape index (NSI), the cell shape index (CSI), the aspect ratio (AR), the cell length (CL), Feret angle and the nuclear positioning. The nuclear positioning is reported as the ratio of the maximum cell length and the maximum cell length protrusion (measured as maximum length from the nucleus).
The coordinate nucleation of F-actin and microtubules in fluorescent images was evaluated using two different Fiji filters on each channel. First, the 8bit images were duplicated and the “north shadow” filter was applied to one. The “convolve” filter and the corresponding color were applied to the duplicated image. The final image is the result of two merged elaborated images (north shadow+convolve colored).