Ix81 inverted fluorescent microscope

Images were acquired using an Olympus IX81 Inverted fluorescent microscope and CellSens Dimension software (Olympus America, Inc., Center Valley, PA). Approximately 15–20 images were collected per cover slip. Neurites of 10–15 neurons per each image were measured according to previously described protocol (Murashov et al., 2005 (link)). The length of the longest neurite for each neuron, as well as the number of neurite branches and number of puncta per neuron were determined using ImageJ software (NIH, Bethesda, MD). Neurite length was measured tracing using only in neurons, which were completely distinguishable from neighboring cells. Axon length was normalized to the average length of the control axons. Synaptic puncta were normalized by neurite length visualized with antibody staining against tubulin or MAP2. The number, size, and colocalization of puncta were measured and analyzed by One-way ANOVA. All analyzes were performed double blind.

Cell proliferation was monitored using the CCK-8 kit (Dojindo, USA). A day before transfection, cells were seeded into 96-well culture plates at a density of 2 × 10³ cells/well. The number of proliferating cells was determined by the absorbance measured at 450 nm using a microplate reader (96-well microplate, Corning) at 0, 24, 48, and 72 h. All experiments were repeated thrice. The EdU incorporation assay was performed using a BeyoClick™ EdU cell proliferation kit with Alexa Fluor 567 (RiboBio, Guangzhou, China). The treated PTC cells were cultured in 96-well plates for 24 h. Then, 100 µL of 50 µM EdU culture medium was added to each well and incubated for 2 h. Each well was further incubated with 4% polyformaldehyde at room temperature for 30 min. Later, 1X Apollo and DAPI staining reagents were added sequentially and incubated at room temperature in a decolorizing shaker for 30 min. The images were captured using an Olympus IX81 inverted fluorescent microscope and CellSens software (Olympus Corporation). All experiments were repeated thrice.

Bone marrow-derived macrophages (BMDM) were obtained from the tibias and femurs of common C57BL/6 adult mice (IL, Cell Biologics, Inc.). The cell culture medium was prepared using Dulbecco’s modified Eagle medium/F12, 10 mM l-glutamine and supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Macrophages were seeded in 12- well or 24-well tissue culture plates at 1 × 10⁶ cells and allowed to adhere for 24 h at 37 °C in humidified atmosphere containing 5% CO₂ prior to experimental studies. Macrophages were polarized to the M1 state by incubating with cell medium containing 5 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF) and stimulated with a combination of 100 ng/ml lipopolysaccharides (LPS) (MO, Sigma-Aldrich) and 20 ng/ml interferon γ (IFNγ) (CA, B&D Bioscience) for 12 h. On the other hand, macrophages were incubated with 2500 U/ml Macrophage colony-stimulating factor (M-CSF) and treated with 20 ng/ml interleukin 4 (IL-4) (CA, eBioscience) for 12 h to activate into the M2 state. The control group was the macrophages incubated with GM-CSF without any cytokine stimulation. The morphology of macrophages was imaged using Olympus IX 81 inverted fluorescent microscope.

For gene expression analysis, granulosa cells isolated from eCG-primed mice were centrifuged at 1,000 g, counted, and seeded at 2 × 10⁴ cells per well in 96-well culture plates previously coated with DMEM/F12 media containing 5% FCS. After overnight culture in standard incubation conditions, the cells were washed and the media was replaced with DMEM/F12 containing a vehicle or 50 mIU/mL of FSH. Separate plates were incubated in atmospheric oxygen (20%), 5% CO₂, and 37°C, while the other samples were placed into a sealed chamber equilibrated with 2% oxygen and 5% CO₂, and placed into the same incubator. After 2 h treatment in these conditions, the cells were harvested and processed for RNA extraction, as described.
For cell morphology analysis, cells were plated in wells pre-coated for 2 h with DMEM/F12 medium containing either 5% FCSor bSA, fibronectin, or semaphorin 7A at the indicated concentrations. After 2 h, the cells were washed with fresh serum-free media and imaged. For cytoskeletal imaging, the cells were fixed in 4% formaldehyde, washed in PBS, and then permeabilized with 0.1% Triton X-100 in PBS for 15 min. The cells were then incubated for 15 min in PBS containing 1X Phalloidin-Alexa-488 (Thermo Fisher) and 1% bSA, then washed in PBS containing Hoechst 33342, and imaged on an Olympus IX81 inverted fluorescent microscope.