Poly d lysine solution

Sicastar®-redF and BNF-Starch-redF (micromod Partikeltechnologie GmbH, Rostock, Germany) are amorphous silica nanoparticles and magnetic bionized nanoferrite nanoparticles, respectively, both 100 nm in size, precoated with amino groups (NH₂), and labeled with a red fluorophore (Sicastar: ex. λ = 569 nm, BNF: ex. λ = 552 nm). The cell-internalization of magnetic BNF nanoparticles was stimulated by a conventional permanent magnet. To prevent nanoparticle aggregation, all nanoparticle associated experiments were performed in serum-free medium Dulbecco’s modified Eagle’s medium with 1% gentamicin^{23 (link),24 (link)}. 50 µg/ml nanoparticle working solutions were prepared at room temperature, protected from light, and coated with 4.5 µg/ml of a poly-D-lysine solution (Sigma-Aldrich, St. Louis, USA) to ensure DNA binding. Nanoparticles were incubated with 50 ng/ml GCSH (Tv1 or Tv*) cDNA for 1 h. Cells were transfected with labeled nanoparticles for 4 h, washed twice with serum-free medium and cultivated up to 24 h in cell culture medium. Following controls were used: 1. Non-transfection control (w/o DNA, w/o nanoparticles); 2. mock control (nanoparticles, w/o cDNA), and 3. GAPDH-control (GAPDH-cDNA, nanoparticles). Transfection efficiency, nanoparticle internalization, and transient expression of GCSH variants were determined by confocal laser-scanning microscopy and flow cytometry.

To assess neurite outgrowth, the PC12 cells were plated at a density of 5 × 10³ cells per well on 35 mm culture dishes coated with poly-d-lysine solution (Sigma-Aldrich). After 12 h, the PC12 cells were incubated with 50 ng/ml NGF2.5S (Millipore) for the indicated times in DMEM medium containing with 1% heat-inactivated HS, 0.5% heat-inactivated FBS, and 100 units/ml penicillin and 100 μg/ml streptomycin. The quantity of neurite bearing cells was determined by counting at least 100 single cells/3 arbitrary positions per dish. A cell was identified to as positive for neurite outgrowth if it had at least a twofold increased cell body diameter. Cells were visualized using a phase-contrast microscope (200x, Nikon TS100, Nikon, Tokyo, Japan).

Cultured primary DRG neurons from thoracic and lumbar (T12–T13, L1–L6) were used for calcium imaging experiments. DRGs were dissected and placed in chilled HBSS solution. The neurons were then digested in collagenase A (1:1; A (Sigma-Aldrich, Cat#10103586001):HBSS (Gibco, Cat#14-170-112)) for 20 min at 37 °C, collagenase D (1:1:10%; D (Sigma-Aldrich, Cat#1188866001):HBSS:papain (Sigma-Aldrich, Cat#10108014001) for 20 min at 37 °C, and then placed in a Trypsin Inhibitor solution (1:1:1; Trypsin (Sigma-Aldrich, Cat#10109886001):BSA: Media) for trituration. Media is DME/F-12 1:1 (1) with 2.50 mM l-Glutamine and 15 mM HEPES buffer (HyClone, Cat#SH30023) supplemented with 10% Fetal Bovine Serum (HyClone, Cat#SH30088.03) and 1% Penicillin Streptomycin (Fisher Scientific, Cat#15070063). After trituration, cells were filtered through a 70 μm cell strainer (Corning, Cat#CLS431751), pelleted, buffer removed, and then resuspended in 200 μL of Media. Plates used were 35 mm Petri dish, 10 mm Microwell, and No. 1.5 cover glass (MatTek Corporation, Cat#P35GC-1.5-C) that were coated in facility with a 2 µg/mL poly-D lysine solution (Sigma-Aldrich, Cat#P0899). Cells were plated in a 200 μL bubble on the center of the plate to rest in an incubator set at 37 °C with 5% CO2 for 2 h before filling the rest of the well with Media. Cells were used the next day for calcium imaging.

Medulloblastoma cell lines DAOY and ONS-76 were cultured in MEM and RPMI 1640 medium (GIBCO), respectively, supplemented with 10% fetal bovine serum (GIBCO), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen), 1.25 µg/ml fungizone (Invitrogen). DAOY Medium was supplemented with 0.1 mM non-essential amino acids and 1 mM sodium pyruvate. Granule cell progenitors (GCP) were purified from mouse cerebella dissected at P7. Following dissociation (30 min, 37 °C) in trypsin/DNAse solution at 37 °C and trituration in DNAse, cells were separated on a density step gradient of 35% and 60% Percoll solution (Sigma). Purified GCPs were further enriched by panning on tissue culture dishes to remove adherent fibroblasts. Non adherent cells were plated at a density of 4 × 10⁶ cells/mL in 12 wells plate coated with a poly D-lysine solution (Sigma) and Matrigel (BD Biosciences), with 12.5 μg/mL SHH (R&D). Cells were grown in Neurobasal medium with B27 supplement, 2 mM glutamine, and 100 U/mL penicillin/streptomycin (all from Invitrogen), linoleic acid-albumin, 0.45% D-glucose, and 16 μg/mL N-acetyl-cysteine, 1% SPITE (all from Sigma-Aldrich). After 1 h plating, GCPs were infected, washed and cultured (37 °C, 5% CO₂) in their medium for 48 h when their RNA was extracted.

Briefly, 1 × 10⁵ HNSCC cells were seeded into SeedEZ^TM scaffold (Lena Bioscience, Atlanta, GA) pre-coated with Poly-D-Lysine solution (Sigma-Aldrich, St. Louis, MO) [15 (link)]. After 10 days of culture, 3D cultures were incubated with free or NP-associated drugs for 12 days. At the endpoint of this experiment, cells in SeedEZ^TM scaffold supplied with complete medium were stained with Texas Red®-X phalloidin (Invitrogen), followed by fluorescence imaging (Zeiss). Cell viability in 3D cultures was quantified by an alamarBlue assay (Bio-Rad).

Satellite cells were sorted based on established methods^{81 (link),82 (link)}. Briefly, entire hindlimb muscles from mice were digested with collagenase II (LS004177, Worthington, 1000 units per 1 ml) for 90 min at 37 °C, the digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% horse serum, heat-inactivated (HIHS, 26050088, Gibco, 1% P/S) before SCs were liberated by treating with Collagenase II (100 units per 1 ml) and Dispase (17105-041, Gibco, 1.1 unit per 1 ml) for 30 min. The suspensions were passed through a 20G needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40-µm cell strainer and sorted by BD FACSAria IV (fluorescence-activated cell sorting) with the selection of the positive GFP fluorescence signal. BD FACSVerse flow cytometer, BD FACSAria Fusion Cell Sorter and BD FACSDiva (Version 8.0.1, BD Biosciences) were used for the acquisition of flow cytometry data. Coverslips and cultural wells were coated with poly-D-lysine solution (p0899, Sigma) at 37 °C for overnight and then coated with extracellular matrix (ECM) (E-1270, Sigma) at 4 °C for at least 6 h. FACS-isolated SCs were seeded in coated wells and cultured in Ham’s F10 medium with 10% HIHS, 5 ng ml⁻¹ β-FGF (PHG0026, Thermo Fisher Scientific) and 1% P/S.