Neurite outgrowth staining kit

IMR-32 (ATCC CCL-127) and SH-SY5Y (ATCC CRL-2266) cell lines were obtained from American Type Culture Collection (ATCC). IMR-32 was maintained in ATCC-formulated Eagle’s Minimum Essential Medium (EMEM, ATCC), and SH-SY5Y was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Waltham, MA, USA). Media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). All the cell lines were maintained at 37 °C in a 5% humidified CO₂ atmosphere in a T-75 flask at a density of 300,000 cells/flask. Cells were passaged when confluency reached approximately 75% using trypsin–EDTA (0.25%) phenol red solution (Gibco, Waltham, MA, USA) for dissociation. For differentiation induction, cells were seeded in a T-25 culture flask at a density of 100,000 cells/flask and treated with ATRA (Sigma Aldrich, Saint Louis, MO, USA) dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich, Saint Louis, MO, USA) at specified concentrations and durations. The ATRA stock solution was prepared at a concentration of 0.5 μg/μL. The differentiation inducer was replenished every 2 days by changing the media. Differentiation extent was evaluated using the Neurite Outgrowth Staining Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines.

To assess neuronal morphology of N2a and HT22 cells, cells were seeded in 24- or 48-well plates to be treated with GEBR32a and/or Aβ_1-42 or to be transfected. N2a cells were treated with DMSO or 0.1–1.0 µM GEBR32a for 48 h (n = 6/condition). HT22 cells were incubated with DMSO (n = 12/condition) or 0.01–1.0 µM GEBR32a (n = 6/condition). In another set of experiments, HT22 cells were treated with 0.5–1.0 µM Aβ_1-42 alone and GEBR32a (1 µM) and Aβ_1-42 (1 µM) and incubated for 24 h (n = 6/condition). For transfection experiments, HT22 cells were cultivated for 48 h upon transfection or exposed to 1 µM Aβ_1-42 for 24 h, one-day post-transfection (n = 9–12/condition). To visualize and quantify neuronal morphology, the Neurite Outgrowth Staining Kit (A15001, Invitrogen) was used to fix and membrane-stain the cells according to the manufacturer’s protocol. Pictures (20 × magnification) were taken with an Olympus IX81 inverted microscope connected to an ORCA-fusion digital CMOS camera (C14440-20UP, Hamamatsu) using the MicroManager open source software [42 (link), 43 (link)]. Per well, three images were captured and used for neurite outgrowth analysis by the NeuronJ plugin for ImageJ [41 (link), 44 (link)]. Average neurite length per condition was normalized against the control conditions (i.e., DMSO-treated or control-transfected cells).

Neurite outgrowth is one of the most important parameters allowing to assess the morphological phenotype of neuronal cells, correlated with their proper functioning and condition. “Neurite Outgrowth Staining Kit” (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA, cat. no. A15001) was used for spectrofluorimetric assessment of neurite outgrowth. This assay was performed on day 3 (this incubation time was chosen based on other assays) to compare the effect of surface modification and NGF administration—neurite outgrowth was measured on collagen and non-coated surfaces, with or without NGF addition.
After removal of the supernatant, the cells were washed with PBS, and the staining solution was added to each well. Cells were incubated for 20 min at 37 °C. After incubation, cell cultures were washed with PBS, a background suppression dye was administered, and a spectrofluorimetric measurement was performed at 485/535 nm (excitation/emission wavelength) using Varioskan LUX microplate reader (Thermo Scientific). Cultures were also analysed under an EVOS FL microscope (Thermo Fisher Scientific) with a fluorescence filter.

For CLSM, neurons were stained using Neurite Outgrowth Staining Kit (Invitrogen, Thermo Fisher). Growth medium was removed and the substrates were treated with 1× dye mixture in Hank's Balanced Salt Solution (HBSS) or Dulbecco's Phosphate-Buffered Saline (DPBS) for 15 min at 37 °C and 5% CO₂. Imaging was performed with a Leica TCS SP8 confocal microscope (excitation at 488 nm and 552 nm wavelength; detection between 494–530 nm and 558–610 nm). Rendering of images to 2D stacks was performed using Leica LAS X software or the Fuji distribution of ImageJ.^{58,59 (link)} The OrientationJ plugin in ImageJ was used for directional analysis of neurite orientation.^{60,61 (link)}

PC-12 cells were seeded in 96-well plates (4 × 10⁴/well) coated with poly-L-lysine. Next day, the cells were treated with compounds (10 µM) in serum-free medium, and neurite outgrowth was measured after 48 h using Neurite Outgrowth Staining Kit (Molecular Probes), in accordance with the manufacturer’s instructions. Fluorescence intensity was measured at 525 nm with excitation at 483 nm for cell viability indicator and at 567 nm with excitation at 554 nm for cell membrane indicator. Results, expressing increased neurite outgrowth, were normalized to cells treated with DMSO.

In an effort to visualise the appearance and adhesion of cells growing on the surface of SF films produced from different treatments, cells were fixed using 4% parafomaldehyde in 1X PBS, 7 days after cell seeding. Then, at least 3 films per treatment, were stained with Neurite Outgrowth Staining Kit according to the manufacturer’s instructions (Molecular probes, Carlsbad, CA, USA). The shape of the cells was monitored via bright orange staining of outer cell membrane surfaces and samples were imaged in a fluorescence microscope (Nikon Diaphot-TMD, Japan).