Extraction solution

NSC 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) according to the manufacturer’s protocol. Briefly, NSC were seeded in the inserted upper chamber. To assess the role of CXCR4 in mediating effects of OPN on migration, we blocked the receptor with AMD3100 at a concentration of 10 μM in half of the wells directly after plating for 1 h as pre-incubation before adding OPN. After 1 h, either 10 μg/ml OPN, 10 % fetal calf serum (FCS) as a positive control, or phosphate-buffered saline as a negative control, were added to the lower chambers of the modified Boyden chamber. After 48 h, the non-migrating cells on the upper side of the inserts were removed with a cotton tip. The migrating cells on the lower side of the inserts were stained with Cell Stain Solution for 10 minutes (Cell Biolabs, Inc.). Subsequently, they were washed with phosphate-buffered saline and allowed to air dry, before they were incubated with the Extraction Solution (Cell Biolabs, Inc.). The optical density of each sample was measured at 560 nm in a plate reader (FLUOstar Omega; BMG LABTECH, Ortenberg, Germany). Mean values ± SEM were established among equally treated samples, and results were expressed as percent of the control.

B16F10 murine melanoma cells were obtained from the ATCC and cultured in RPMI-1640 medium supplemented with 10% FBS. Cells were seeded at 2.0 × 10⁵ into an insert well (6.5 mm diameter Transwell with 8 μm pores; Corning, Corning, NY, USA) in 200 μL of RPMI-1640 medium supplemented with 1% FBS. In the bottom chamber, 600 μL of RPMI-1640 medium with 1% FBS with or without test proteins was added. Cells were cultured for 20 h and fixed with 4% paraformaldehyde in PBS for 30 min. Cells attached to the bottom side of the membranes were stained with 0.4% crystal violet in 20% methanol and extracted with 200 μL of Extraction Solution (Cell Biolabs, San Diego, CA, USA). The absorbance at 560 nm was measured with an ARVO MX (PerkinElmer, Akron, OH, USA).

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.

Cell migration was analyzed via a trans-well migration assay using a modified Boyden chamber (CytoSelect^TM 24-Well Cell Migration Assay, 8 μM pore size, Cell Biolabs, Inc., San Diego, USA), following the manufacturer’s protocol. Briefly, freshly collected cells (50,000 microglia) were dissolved in a serum-free culture medium within the inserted upper chamber. As a chemo-attractive stimulus, a medium containing 10% FBS was added to the lower chamber. Either 10 μM or 30 μM of ZD7288 or XE-991, respectively, were added to the upper chamber of the modified Boyden chamber. Untreated cells served as a 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) on a plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany). Mean values +/− SEM were established among equally treated samples. Each experiment was conducted in triplicate.