Fv 1000 point scanning confocal microscope

Cell viability was assessed using a LIVE/DEAD® viability/cytotoxicity assay kit (Invitrogen, Bio-science, Ireland). Briefly, constructs were incubated in live/dead solution containing 2μM calcein AM (intact cell membrane) and 4μM ethidium homodimer-1 (disrupted cell membrane; both from Cambridge Bioscience, Cambridge, UK). Sections were imaged with an Olympus FV-1000 Point-Scanning Confocal Microscope at 515 and 615 nm channels and analysed using FV10-ASW 2.0 Viewer software. Semi-quantitative analysis of LIVE and DEAD fractions were determined using IMAGEJ software (National Institutes of Health, Bethesda, Maryland, USA) and averaged over N=3 samples.

Expanded hMSCs (passage 2) were seeded on the CT and HT scaffolds to ensure cells were capable of migrating throughout the construct and that a homogenous distribution could be achieved. Cell viability was assessed after 24 h using a LIVE/DEAD Ò viability/cytotoxicity assay kit (Invitrogen, Bio-science, Ireland). Briefly, constructs were cut in half, washed in PBS followed by incubation in PBS containing 2 lM calcein AM (green fluorescence of membrane for live cells) and 4 lM ethidium homodimer-1 (red fluorescence of DNA for dead cells; both from Cambridge Bioscience, UK). Sections were again washed in PBS, imaged at magnification Â10 with an Olympus FV-1000 Point-Scanning Confocal Microscope (Southend-on-Sea, UK) at 515 and 615 nm channels and analysed using FV10-ASW 2.0 Viewer software. Z-stack images of 30 scans every 10 lm in the cross-section of the scaffold were acquired for a total of 300 lm depth.

Calcium deposition within the scaffolds was measured using the Calcium LiquiColor ® Test (Stanbio Laboratories) according to the manufacturer's protocol. Briefly, a cell lysate was prepared using 0.5 mL of Triton X-100 and 0.5 mL trichloroacetic acid. Samples were incubated for 60 min and, then, centrifuged. The supernatant was used to calculate calcium levels. Next, 10 μL of each sample and assay standard were added to a 96-well plate and 200 μL of the working solution were added. The plate was analysed on a microplate reader (Biotek, Synergy HT, Swindon, UK) at an absorbance of 550 nm, as previously described (Shor et al., 2009) (link). The calcium incubation, the constructs (n = 4) were rinsed again in PBS and imaged with Olympus FV1000 pointscanning confocal microscope at 488 and 543 nm.

Cell metabolic activity was evaluated using AlamarBlueTM (BioSciences, Dun Laoghaire, Ireland), a non-endpoint, non-toxic assay, after 3 and 7 days of nHA and PEI-mediated gene delivery in monolayer, and after 1 and 7 days in the 3D gene-activated alginate hydrogel system. 10% AlamarBlueTM in 1 ml of standard culture media was used for the assay. All samples were incubated for 4 hours at 37 ⁰C. After the incubation time, 200 μl of the supernatant was plated in triplicate into a 96-well plate, absorbance was read at 570 nm and 600 nm and its reduction was translated to cell activity relative to the untransfected control.
For nHA-pLuc transfected groups, cell viability was assessed using LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Invitrogen, Bio-science, Ireland). Images were taking by confocal microscopy using an Olympus FV-1000 Point-Scanning Confocal Microscope (Southon-Sea, UK) at 488 and 543 nm channels and analysed using FV10-ASW 2.0 Viewer software, and cell viability was calculated using ImageJ software.
DNA analysis was performed by digesting the samples with papain (125 µg/ml, pH = 6.5) in 0.1 M sodium acetate, 5 nM L-cysteine HCl, and 0.05 M EDTA (Sigma-Aldrich, Ireland) at 60 ⁰C under constant rotation for 18 hours. DNA content was quantified using the Hoescht 33258 dyebased DNA QF kit and standard curve (Sigma-Aldrich, Ireland).