Cytoselect 24 well cell migration assay

RPE cell migration was performed using SPLScar Scratchers (24-well lid, SPL Life Sciences, Pocheon, Korea). All procedures were performed according to the manufacturer’s instructions. Straight-line scratches across RPE cells were made using SPLScar Scratchers and then treated with Nano-GO (20 and 40 μg/mL) for 24 h and 48 h, photographed from the straight-line scratch across RPE cells under a stereomicroscope (SMZ800, Nikon Corporation, Tokyo, Japan). Images were captured using an attached IMT i-Solution CAM 3 (IMT iSolution Inc., Vancouver, BC, Canada). The RPE cell migration assay was performed using the CytoSelect 24-Well Cell Migration Assay (Cell Biolabs, San Diego, CA, USA). All procedures were performed according to the manufacturer’s instructions. RPE cells were treated with Nano-GO (20 and 40 mg/mL) for 24 h. Migratory cells were fixed and stained with cell stain solution, and the whole area was photographed (upper chamber) under a stereomicroscope (SMZ800, Nikon Corporation, Tokyo, Japan). Images were captured using an attached IMT i-Solution CAM 3 (IMT iSolution Inc., Vancouver, BC, Canada). Subsequently, the stained cells were extracted with an extraction solution and then measured at 560 nm using an Omega Plate Reader (BMG Labtech, Ortenberg, Germany).

Since Raji cells express CXCR5 [13 (link)], which plays a role in B-cell migration, we assayed the migration of a cell suspension of Raji cells transduced with TAGLN2-specific siRNA (OriGene) or a scrambled negative control siRNA for 24 h. The cells, suspended in RPMI1640 containing 1% FBS, were loaded into the upper chamber of the CytoSelect™ 24-Well Cell Migration Assay (Cell Biolabs, Inc. San Diego, CA, USA) and were cultured for 4 h. Cells migrating into the lower chamber, which contained RPMI1640 plus 1% FBS with 750 ng/mL of recombinant CXCL13 (CXCR5 ligand; Biolegend, San Diego, CA, USA) were lysed and quantified using the CyQuant GR Fluorescent Dye and a fluorescence plate reader at 480 nm/520 nm (Infinite F200, TECAN, Tecan Japan, Kanagawa).

The cell migration was analyzed via a trans-well migration assay using a modified Boyden chamber (CytoSelect™ 24-Well Cell Migration Assay, 8-μM pore size, Cell Biolabs, Inc., San Diego, USA), following the manufacturer’s protocol. Briefly, freshly collected cells were dissolved in serum-free culture medium within the inserted upper chamber (50,000 NSCs or 10,000 microglia). For microglia experiments, either 10 ng/ml LPS or 50 ng/ml recombinant rat IL4 were added to the upper chambers of the modified Boyden chamber. In NSC experiments, LPS 10 ng/ml, recombinant rat IL4 (50 ng/ml), or conditioned medium (of microglia pre-treated with LPS, IL4, or LPS + IL4) were filled into the lower chamber of the plate. Untreated cells served as a control, and 10% FBS-containing medium in the lower chamber served as a positive control. After 24 h, migrated cells on the lower side of the inserts were stained with crystal violet, extracted (extraction solution, Cell Biolabs, Inc., San Diego, USA), and quantified by photometrical detection (560 nm) in a plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany). Mean values ± SEM were established among equally treated samples. Each experiment was conducted in triplicate.

In vitro DMSC migration was determined using Millicell 24-well transwell Boyden chambers with 8 μm pore polycarbonate membranes (Merck Millipore, Spain) as we previously described (Vegh et al., 2013 (link); Paris et al., 2016 (link); Paris et al., 2017 (link)). Briefly, 2 × 10⁵ DMSC in 100 μl of migration media (DMEM supplemented with 55 μM b-mercaptoethanol, 1% non-essential amino acids and without FBS) were seeded in the upper chamber of the transwell system and 5 × 10⁴ suburethral tissue cells were seeded on the well below. Migration medium without cells was used as a negative control. The migration was assessed at 24 h by the CytoSelect 24-Well Cell Migration Assay (8 μm, Colorimetric, Cell Biolabs, bioNova, Madrid, Spain) following the manufacturer´s instructions. Migratory cells were stained with the Cell Stain Solution and the color was subsequently extracted with the Extraction Solution, and quantified by absorbance at 560 nm using an Enspire 2300 plate reader (PerkinElmer, Tres Cantos, Madrid, Spain). All experiments were done in triplicate.