Dulbecco phosphate buffered saline (dpbs)

This study investigated the binding of CNGRC cyclopeptide, and the control cyclopeptides (CARGC, CDGRC) onto living cell lines by TR-NOESY, as previously described [43 (link)]. Briefly, the cells were detached in DPBS (Lonza) with 2 mM EDTA, and transferred into a 3 mm NMR tube at the concentration of 8–10 × 10⁶ cells /200 μL of deuterated DPBS supplemented with 3 mM MgCl₂. The NMR experiments were performed at 37 °C, for 2 h, at a field strength of 600 MHz. The cell suspension homogeneity and >75% cell viability were achieved throughout each experiment. To discriminate binder from non-binder cells, the different NOE build-up rate, i.e., the time required to achieve maximum NOE intensity, was exploited, and the experiments were performed at short (100 ms) and long (600 ms) mixing times. The ligands interacting with surface receptors have a fast NOE build-up, with the maximum NOE intensity (cross-peaks with the same sign as the diagonal) at short mixing time (100 ms), and at longer mixing time (600 ms) the signal has already decayed. Conversely, the negative control peptides that do not bind and therefore have a slower NOE build-up curve, have no (or very weakly positive) NOE effect at 100 ms, whereas they develop the strongest NOE intensity (cross-peaks with opposite sign with respect to the diagonal) at longer mixing time (600 ms).

hBM-MSCs were seeded (2 × 10⁵cells/flask) and grown on a T-25 bottle to achieve 70% confluence and then labeled with a suitable stain (Molday ION Rhodamine B™, CellTracker™ Green CMFDA or Stemgent® eEGFP) according to the procedures described above. Labeled cells were stripped from the bottom of the culture vessel with trypsin, centrifuged (3 min 1000g), and counted. hBM-MSCs were then plated in the number of 1 × 10³ on 24-well in MSCGM™ mesenchymal stem cell growth medium. Four hours later, the culture medium was exchanged to StemPro Osteogenesis Differentiation Kit, where the cells were cultured for another 3 weeks at 37 °C, 5% CO₂. The culture medium was replaced every third day. After 21 days, cells were rinsed with DPBS (Lonza) and fixed in 4% PFA for 30 min. Cells were then washed twice with distilled water and stained with 2% Alizarin Red Solution (Millipore) for 3 min. Excess of the dye was eliminated by three times distilled water rinsing. Evaluation of morphology and fluorescence of cells was performed using the Axiovert.A1 Zeiss fluorescence microscope in the Zen blue software.

Human gingival fibroblasts (Innoprot, Bizkaia, Spain) were cultured in tissue flasks of polystyrene in a CO₂ incubator in a Nuaire US Autoflow NU-4750-E (Nuaire, Plymouth, Minnesota, USA) at 37 °C in humidified 5% carbon dioxide (CO₂) with a 95% air atmosphere for 1–2 weeks. Dulbecco’s modified Eagle medium (DMEM, Biowest, Nuaillé, France) was supplemented with 10% FBS (Biowest, Nuaillé, France) and 1% glutamine–penicillin–streptomycin (Biowest, Nuaillé, France). The medium was changed every 48 h, and the cells were subcultured regularly upon reaching 80% confluence. Later, the cells were passaged after trypsinization using 0.25% trypsin (Biowest, Nuaillé, France) and Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS, Lonza, Basel, Switzerland), which was previously tempered in a water bath at 37 °C. Cellular growth, adhesion, and proliferation were monitored with an Olympus CKX41SF2 microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Cultured HGFs from the second to eighth passages were used for the experiments.

After storage in liquid nitrogen for 2–5 days, vials were thawed with gentle agitation at 35 °C in a water bath until an ice ball was no longer present. Immediately post thaw, an equal volume of DPBS was added to the cell suspension. Five minutes later, the cell suspension was collected and added dropwise to 20 ml DPBS (Lonza). This thawing method was defined in a pilot project to this experiment where we determined the importance of avoiding osmotic shock in the 95/5 formulations (Additional file 1). A 100 μl aliquot of thawed cell suspension in DPBS was used for a total cell count and viability using fluorescein diacetate (67.57 mg/ml) and propidium iodide (1.35 mg/ml) in DPBS. Counting cell suspensions were plated on a Nebauer hemocytometer and visualized by fluorescence microscopy (Olympus, Center Valley, PA, USA). The live (green) and dead (red) cells were counted. A total of 10 squares were counted per sample. The cell suspension was carefully mixed by pipetting and then centrifuged to pellet the cells for removal of freezing solutions (300 × g, 5 minutes, 4 °C, 7 brake).