Page 84 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 4
respect to the start of insonification) was determined at 10, 20, 40, 80, and 160 kPa PNP. To check the effect of insonification of HUVECs on SPIO uptake in the absence of tMBs, the OptiCells were insonified at 40, 80, and 160 kPa PNP for 30 s, while SPIO was added 5 min prior to insonification (n = 2). All other experiments were repeated three times. From these three datasets the average and standard deviation are plotted.
SPIO labeling
After the treatment described above (see also Fig 1B), cells were rinsed three times with phosphate-buffer saline (PBS; Invitrogen, Groningen, the Netherlands) to remove non-internalized SPIO. Then, cells were fixated with 4% formaldehyde (Sigma-Aldrich, Zwijndrecht, the Netherlands) for 10 min. After fixation, the cells were washed three times with PBS and then incubated with Prussian Blue solution for 30 min (aqueous solution of 10% hydrochloric acid (Sigma-Aldrich) and 5% potassium ferrocyanide (Sigma-Aldrich)) to assess the SPIO-labeling [52]. Next, the cells were washed three times with PBS and the nuclei were stained with 0.1% nuclear fast red solution (Sigma-Aldrich). Thereafter the OptiCells were dried for 48 h and microscopically examined using a microscope (Olympus, Zoeterwoude, the Netherlands) equipped with 20× Plan (NA 0.4) objective (Olympus) and a color camera (Axiocam MRc, Carl Zeiss, Germany). SPIO uptake was assessed by manually counting Prussian Blue positive cells among ~500 cells (acquired in 5 fields of view) located within a circle of 6 mm diameter around the center point of each insonified area. A cell was counted as SPIO positive when it contained one or more Prussian Blue stained iron particles.
Cell viability assay
For each SPIO uptake measurement, cell viability was determined in triplicate by calcein-AM and propidium iodide (PI) assays in parallel. Cells were treated with SPIO, tMB and ultrasound as described before (see also Fig 1). Within 3 to 4 min after the US treatment of all subsections of the Opticells, HUVECs were incubated at 37 °C, 5% CO2. Thirty min before assessing the cell viability, calcein-AM was added to the OptiCell chamber (C3100MP; Invitrogen; 0.25 μM final concentration from a 1 mM stock prepared in DMSO (Sigma- Aldrich)) and incubated for 30 min under the same conditions. Thereafter PI (P4864, Sigma-Aldrich, 25 μg/ml final concentration) and Hoechst
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