Ix2 ucb

Absorptive enterocyte monolayers were treated with vehicle or 20 umol/L etomoxir and exposed apically to B-C12 or B-C16 for 6 hours. After 6 hours of apical B-C12 or B-C16 exposure, absorptive enterocyte monolayers were rinsed with fresh DM and fixed in 40% glyoxal solution for 20 minutes. After fixation, absorptive enterocyte monolayers were rinsed with 1× DPBS. Next, the Transwell membrane containing the fixed absorptive enterocyte monolayers were removed from the Transwell inserts and placed on glass slides. Absorptive enterocyte monolayers then were overlaid with mounting media and covered with a glass coverslip. Fluorescent images used for quantification were taken at 20× magnification on an Olympus IX2-UCB (Olympus, Shinjuku City, Tokyo, Japan) microscope. Representative fluorescent images of absorptive enterocyte monolayers in Figure 5 were taken at 40× magnification on a Keyence BZ-X810 microscope (Keyance, Osaka, Japan).

Flow cytometric analyses of the transfected HEK293, CHO, and A549 cells was performed to detect eGFP and CD39 expression. Therefore, 24 h, 72 h, 120 h, and 168 h after transfection, CD39-fluorescein isothiocyanate (FITC) antibody (Abcam, U. K.: Clone A1) was used to detect CD39 expression using a FACS Calibur (BD Bioscience, U. S. A.). A total of 10,000 events were acquired in each sample. For the analysis of eGFP expression, no antibody was necessary.
The eGFP expression of CHO cells was additionally detected via fluorescence microscopy 24 h after transfection using a fluorescence microscope (IX2UCB, Olympus, Japan). The fluorescence intensity was analyzed by measuring the grey scale of the images using ImageJ.

Cell migration was evaluated by in vitro wound healing assay. Cells were grown to confluence in 6-well plates. Cell monolayers were scraped with a sterile micropipette tip and treated with different concentrations of FH535. The wound area was photographed by microscope (Olympus IX2-UCB) before and 24 hours after the treatment. The wound widths were measured using ImageJ.
Transwell invasion assay was carried out using 24-well Transwell chamber with an 8 µm pore size polycarbonate filter membrane (Corning). Before the assay, Matrigel (1:10 dilution, BD Biosciences) was coated in the upper chamber overnight. 1×10⁵ cells in 200 µl RPMI 1640 with 1% FBS were incubated in the upper chamber, 900µl RPMI 1640 with 10% FBS were added in the lower chamber. After incubation for 36 hours, invaded cells were fixed by 4% PFA and stained with 0.1% crystal violet, then photographed by microscope. The results were presented as counted cells per field at 400× magnification.

5–6 weeks old Sprague Dawlye (SD) male rats were used to create critical-sized cranial bone defect model for in vivo study. Care, housing and surgery procedure use of laboratory animals followed the protocol approved by the Animal Ethics Committee of Wenzhou Medical University (China). An 8 mm defect was made and sterilized BG-XLS, BG-XLS/GelMA-DFO and BG-XLS-BMP2 scaffolds were implanted into the cranial defects. 1 mg/mL of bone morphogenetic protein-2 (BMP2) (Peprotech, Rocky Hill, NJ, USA) solution was mixed with collagen solution (2.5 mg/mL, Bedford, MA, USA), and 1 M NaOH solution was used to adjust the pH value to 7.4. Finally, 10 μL of the collagen/BMP2 solution with 1 μg BMP2 was then incorporated into the BG-XLS scaffolds. The BG-XLS-BMP2 group was chosen as the positive control. All SD rats were euthanized after 8 weeks of implantation, followed by being fixed with 10% formalin for 2 days, then immersed in 70% ethanol for radiographic (Bruker, Billerica, MA, USA) and micro-CT (Skyscan 1076, Bruker, USA) analysis. After the μCT analysis, the decalcification process of samples was carried out in a 10% EDTA (pH = 7.4) solution for 3 weeks and the samples were then imbedded in paraffin for histological analysis. The tissue slices were cut with the thickness of 5 μm and stained with Hematoxylin and Eosin (H&E) and observed under microscope (IX 2-UCB, Olympus).