Evos fluorescent inverted microscope

Oxidative stress analysis in Fe₃O₄ and α-Fe₂O₃/C exposed embryos was done by staining of treated and untreated embryos using 1.25 mg/L 2′,7′-dichlorofluorescin diacetate (H₂DCFDA) dye for 20 min in dark [12 (link)]. The embryos were exposed as mentioned in protocol for biocompatibility assays. To extrapolate the extra stain, washing was performed with HF buffer. Analysis was performed using EVOS inverted fluorescent microscope (ThermoScientific). Data analysis and presentation was done with the help of ImageJ. All the experiments were performed in triplicates and statistical analysis was performed using GraphPad prism 6.

The migration area was calculated by performing the wound healing assay. Briefly, cells at a density of 30 × 10³ cells/cm² in a 48 well plate were plated and allowed to adhere for 24 h. A scratch was applied with a 200 µL pipette tip and the width was measured as the baseline. Cells were incubated with transwell inserts (0.4 µm pore size, Greiner Bio-One, United Kingdom) containing EV-functionalised hydrogels for 3 days. Cells cultured without hydrogels were used as the control. The area of wound closure from day 0 was assessed using fluorescent microscopy. Briefly, cells were labelled with Calcein-AM (1 μg/ml in PBS, Sigma-Aldrich, United Kingdom) in the dark for 30 min. Samples were observed under an EVOS fluorescent inverted microscope (Thermo Scientific, United Kingdom).

Osteoblasts were seeded at 3 × 10³ cells/cm² within a 96‐well plate with basal medium and incubated for 24 h. Media was replaced with fresh basal medium supplemented with/without TSA (Sigma‐Aldrich, UK) (5, 10, 20, 50, 100 nM) and incubated for 1, 3 and 7 days. At each time point, AlamarBlue reagent (Thermo Scientific, UK) was added and incubated for 4 h at 37°C. Fluorescence readings was acquired using a SPARK spectrophotometer (TECAN, CH) at an excitation/emission wavelength of 540/590 nm, respectively. Employing the same protocol, the osteoblast morphology via calcein‐AM staining (Sigma‐Aldrich, UK) after 3 days of culture, observed under an EVOS fluorescent inverted microscope (Thermo Scientific, UK).

The viability and proliferation of hBMSCs (5 × 10⁵ cell/ml) within the EV-functionalised 65/35 composite hydrogels was assessed via live/dead staining. Briefly, hBMSCs (5 × 10⁵ cell/ml) were mixed with the EV-functionalised hydrogel (0, 50, or 100 µg/ml of EV protein) prior to gelation. Following sol-gel transition, hydrogels were culture in basal medium. At day 7, cell-laden hydrogels were incubated with Calcein-AM (1 μg/ml in PBS, Sigma-Aldrich, United Kingdom), and Propidium iodide (1 μg/ml in PBS, Sigma-Aldrich, United Kingdom) in the dark for 30 min. Samples were observed under an EVOS fluorescent inverted microscope (Thermo Scientific, United Kingdom). The proliferation of cell-laden EV-hydrogels was assessed via quantifying DNA content following culture in basal medium for 7 days.
The capacity of EV-functionalised hydrogels to stimulate encapsulated hBMSCs (1 × 10⁶ cell/ml) osteogenic differentiation and mineralisation was evaluated after culture in osteogenic medium for 3 weeks. Osteogenic differentiation was assessed by quantifying alkaline phosphatase activity, collagen production and mineral deposition, detailed below. Cell-free hydrogels of each composition were cultured in the same conditions as described above and used as an acellular control for the following analysis.

The influence of hydrogel composition on proliferation was assessed via quantification of DNA content. Briefly, MC3T3s were mixed at low density (5 × 10⁵ cell/ml) in the hydrogel prior to gelation. Following sol-gel transition, cell-laden hydrogels were cultured in basal medium for 2 weeks with media changes every 3 days. The cellular morphology was assessed at day 3 by incubated cell-laden hydrogels with Calcein-AM (1 μg/ml in PBS, Sigma-Aldrich, United Kingdom) and Propidium iodide (1 μg/ml in PBS, Sigma-Aldrich, United Kingdom) in the dark for 30 min. Samples were observed under an EVOS fluorescent inverted microscope (Thermo Scientific, United Kingdom). The DNA content was assessed using PicoGreen (Life Technologies, United Kingdom) according to the manufacturer’s protocol.
To evaluate the hydrogel effect on osteoinduction, MC3T3s were mixed at high density (1 × 10⁶ cell/ml) in the hydrogel prior to gelation. Following sol-gel transition, hydrogels were incubated in basal medium for 24 h. The media was replaced with osteogenic medium and gels were cultured for 2 weeks, with media changes occurring every 3 days.

hBMSCs were seeded at 3 × 10³ cells/cm² within a 96-well plate with a basal medium and incubated for 24 h. The medium was replaced with a fresh basal medium supplemented with/without AZT (Sigma-Aldrich, Gillingham, UK) (5, 10, 20, 50 µM) or DFO (Sigma-Aldrich, Gillingham, UK) (5, 10, 20, 50 µM), and incubated for 1, 3 and 7 days. At each time point, AlamarBlue reagent (Thermo Scientific, Paisley, UK) was added and incubated for 4 h at 37 °C. Fluorescence readings were acquired using a SPARK spectrophotometer (TECAN, Männedorf, Switzerland) at an excitation/emission wavelength of 540/590 nm, respectively. Employing the same protocol, the cell morphology was assessed via calcein-AM staining (Sigma-Aldrich, Gillingham, UK) after 3 days of culture, and observed under an EVOS fluorescent inverted microscope (Thermo Scientific, Paisley, UK).