Laminin

As previously reported (Shi et al., 2012 (link)), hiPSCs were differentiated in N3 culture medium which consists of DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), Neurobasal (1x, Thermo Fisher Scientific, 21103049), N-2 Supplement (1%, Thermo Fisher Scientific, 17502048), B-27 Supplement (2%, Thermo Fisher Scientific, 17504044), GlutaMax (1%, Thermo Fisher Scientific, 35050061), MEM NEAA (1%, Thermo Fisher Scientific, 11140050), and human recombinant insulin (2.5 µg/mL, Thermo Fisher Scientific, 12585014). From Day 1 to 11, the N3 media was further supplemented with two factors: SB-431542 (5 µM, Tocris, 1614) and LDN-193189 (100 nM, Stemgent, 04007402). At Day 12, the differentiating cells were dissociated with Cell Dissociation Solution (1x, Sigma-Aldrich, C5914), passaged onto Poly-D-Lysine (50 µg/mL, Sigma-Aldrich, P1024) and Laminin (5 µg/mL, Roche, Basel, Switzerland; 11243217001) coated plates, and cultured in N3 media without factors until Day 22 when they were passaged again. Between Day one and Day 22, media changes were performed daily. However, after Day 22, cells were exposed to media changes every other day with N3 media without factors.

Dissociated hippocampal cultures were prepared from embryonic day 18 (E18) rat brains of both genders, as described in [74 (link)]. Dissociated neurons were plated on Ø18-mm coverslips coated with poly-L-lysine (37.5 μg/ml, Sigma-Aldrich) and laminin (1.25 μg/ml, Roche Diagnostics) at a density of 100,000 neurons per well. Neurons were grown in Neurobasal medium (NB) supplemented with 1% penicillin and streptomycin (pen/strep), 2% B27, and 0.5 mM L-glutamine (all from Gibco) (NB-complete medium) at 37°C in 5% CO_2. From DIV 1 onward, medium was refreshed weekly by replacing half of the medium with Brainphys neuronal medium supplemented with 2% NeuroCult SM1 neuronal supplement (STEMCELL Technologies) and 1% pen/strep (BP-complete medium).

Dissociated hippocampal cultures were prepared from embryonic day (E)18 rat brains of both genders (Kapitein et al., 2010b (link)). Dissociated neurons were plated on Ø18-mm coverslips coated with poly-L-lysine (37.5 μg/ml, Sigma-Aldrich) and laminin (1.25 μg/ml, Roche Diagnostics) at a density of 100,000 neurons per well, in Neurobasal medium (NB), supplemented with 1% penicillin and streptomycin (pen/strep), 2% B27, and 0.5 mm L-glutamine (all from Invitrogen; NB-complete medium) at 37°C in 5% CO_2. From day in vitro (DIV) 1 onwards, medium was refreshed every 7 d by replacing half of the medium with Brainphys neuronal medium supplemented with 2% NeuroCult SM1 neuronal supplement (Stem Cell Technologies) and 1% pen/strep (BP-complete medium).

Primary hippocampal neurons cultures were prepared from embryonic day 18 rat brains (both genders). Cells were plated on coverslips coated with poly-l-lysine (37.5 μg/ml; Sigma-Aldrich) and laminin (1.25 μg/ml; Roche Diagnostics) at a density of 100,000 per well of 12-wells plates. Neurons were first cultured in neurobasal medium (NB) supplemented with 2% B27 (GIBCO), 0.5 mM glutamine (GIBCO), 15.6 μM glutamate (Sigma-Aldrich), and 1% penicillin/streptomycin (GIBCO) at 37°C in 5% CO₂. From DIV1 onward, half of the medium was refreshed weekly with BrainPhys medium supplemented with 2% NeuroCult SM1 neuronal supplement (STEMCELL Technologies) and 1% penicillin/streptomycin. Hippocampal neurons were transfected using Lipofectamine 2000 (Invitrogen). Briefly, DNA (1.8 μg per well, of a 12-well plate) was mixed with 3.3 μl of Lipofectamine 2000 in 200 μl of NB, incubated for 20 min, and then added to the neurons in neurobasal at 37°C in 5% CO₂ for 1 hour. Then, neurons were washed with neurobasal and transferred back to their original medium. Transfection of knock-in constructs was performed at DIV3.

DRGs from miR-21–cKO mice and their littermate controls were collected and placed into Ham’s F-12 Nutrient Mixture (Gibco). DRGs were then dissociated using 3 mg/mL Dispase (Roche), 0.1% collagenase (Sigma-Aldrich), and 200 U/mL DNase I (Sigma-Aldrich) in F-12 medium (Gibco). Thereafter, DRGs were triturated, and cell suspensions centrifuged at 300g for 6 minutes. Pellets were resuspended in fresh DRG medium and plated on glass coverslips precoated with poly-L-ornithine (100 μg/mL; Sigma-Aldrich) and laminin (40 μg/mL; Roche). Cultures (10,000 cells/well) were incubated at 37°C for 24 hours, and then stimulated with vehicle or capsaicin (1 μM) for 3 hours. Culture media were removed, and CCL2 was measured using ELISA (R&D Systems, DY479-05) in DRG neuronal culture media, according to the manufacturer’s instructions.
Transmigration assay was performed using cell culture inserts (Transwell plate, Costar) with 8-μm porous polycarbonate filters. DRG neurons (10,000) were cultured in 300 μL F-12/10% FBS in the lower compartment for 24 hours, and then 40,000 WT BMDMs treated with vehicle or CCR2 antagonist (1 μM; Merck) were added to the upper filter. DRG neurons were then stimulated with capsaicin (1 μM) for 3 hours.