Supplementary MaterialsAdditional file 1: Number S1. and expanded in standard tradition medium consisting of DMEM supplemented with 10% (v/v) FBS, 1% (v/v) non-essential amino acids, 1?mM?l-glutamine, 1?mM pyruvate and 1% penicillin/streptomycin. Cells were passaged using trypsin/EDTA. Mouse embryonic stem cell tradition Mouse embryonic stem cells (ESC) were cultured in DMEM supplemented with 1.7?mM?l-glutamine, 0.1?mM -mercaptoethanol, 5?ng/ml mouse leukaemia inhibitory element (LIF), 10% (v/v) FBS, 1% (v/v) non-essential amino acids, 1?mM pyruvate and 1% penicillin/streptomycin (stock 10,000?U/ml) without a feeder coating. Cells were dissociated by 0.05% trypsin/EDTA. Cell labelling with magnetic particles Cells were seeded at 40% confluency and cultivated to 80% confluency before labelling. Fluorescently tagged magnetic particles of 500?nm and 1000?nm (ScreenMAG-Silanol, Chemicell, Germany) were used for cell labelling. Labelling of cell monolayers was performed as explained previously [38, 46]. Briefly, adherent cell populations were incubated with MPs (10?g Fe/ml standard dose or 25?g Fe/ml for fully confluent cultures) in medium for 24?h. The next day, cells were thoroughly washed with PBS in order to remove excessive particles attached to the cell surface or flask. For suspension cell labelling, MSC, CMC and ReN were evenly suspended in 7?ml growth medium without serum and MPs were added at 70?g Fe of particles per 1??106 cells. Cells were agitated at 60 RPM for 3 h and labelled suspensions were then centrifuged to remove excessive particles before plating out or direct circulation cytometry after fixation with 4% ice-cold paraformaldehyde (PFA) (VWR, UK). Particle labelling assessment To measure particle uptake by circulation cytometry, cells were harvested, centrifuged at 200? for 5?min and re-suspended in PBS prior to analysis. Fixed samples from suspension labelling were analysed in PBS immediately following PFA fixation. Labelled and unlabelled populations were compared to evaluate the percentage uptake based Amlexanox on fluorescent intensity. Analysis was performed on a Beckman Coulter FC500 8HT Circulation Cytometer (Beckman Coulter, USA) with WEASEL (WEHI, Australia), using unlabelled cells as settings to evaluate improved fluorescence. Particle uptake was further evaluated visually using fluorescence and super-resolution microscopy. Adherent cells from monolayer cultures or plated out after suspension culture were fixed with 4% PFA and stained using FITC-labelled Phalloidin (Existence Technologies, USA) according to the manufacturers instructions [38, 47], following permeabilisation with 0.1% Triton X-100 for 5?min. Slides were incubated inside a dark covered container at space temp for 15?min, and then washed twice with PBS and counterstained with Hoechst 33342 (Sigma Aldrich, UK). Cells were then imaged using the Operetta Large Content Analysis System (Perkin Elmer, USA). For super-resolution microscopy, CMC were seeded in Matrigel-coated glass-bottom tradition dishes (MatTek Corporation, USA) and remaining to attach and beat for 3?days. Cells were then labelled with 10?g Fe/ml for 24?h, washed three times with PBS and fixed with PFA. MSC osteogenic differentiation MSC were seeded at 5??103 cells/cm2 and the medium was then changed every 3?days for 14?days with either control medium or osteogenic induction medium containing DMEM supplemented with 100?nM dexamethasone, 0.05?mM?l-ascorbic acid-2-phosphate and 10?mM -glycerophosphate. Mineralised nodules were recognized using Von Kossa staining . Cells were fixed at space temp for 15?min in 4% PFA, washed three times with dH2O and incubated with 1% metallic nitrate in dH2O (Sigma Aldrich) under a UV light for 15?min. Samples were washed three times with dH2O, incubated for 5?min with 2.5% sodium thiosulfate solution (Sigma Aldrich), washed again with dH2O and imaged Rabbit Polyclonal to GPR126 using an eclipse TS100 inverted microscope (Nikon, Japan). ReN differentiation Cells were seeded at 10,000 cells/well onto laminin-coated 96-well plates (BD Biosciences) and expanded for 2 days in growth medium before initiating differentiation using ReN tradition medium without growth factors . After 7?days of differentiation, cells were fixed in 4% paraformaldehyde (PFA) for immunocytochemistry analysis. Cell viability assessment Viability was evaluated using the resazurin metabolic assay, using a operating solution consisting of 10% (v/v) Presto Blue stock solution prepared according to the manufacturers instructions. After Amlexanox 45-min incubation, the fluorescent transmission of 100?l samples was measured at 535?nm excitation and 615?nm emission in triplicate, using an Infinite 200 PRO plate reader and i-control software Amlexanox (Tecan, Switzerland). The positive control consisted of unlabelled cells which remained in monolayers for the duration of the labelling time period, and the bad Amlexanox control consisted of cells exposed to 70% methanol in H2O fixative for 10?min followed by 3 PBS washing. HypoThermosol refrigerated storage of cells MSC, CMC and ReN at full.