Fibronectin coated 24 well plates

Fibronectin-coated 24-well plates (Corning, New York, NY) were seeded with, 1 × 10⁴ cells/well and incubated for 15 min, after which the non-adherent cells were washed away and the adherent cells were fixed in 4% paraformaldehyde, stained with crystal violet and counted (5 random 100× fields per well). Three independent experiments were performed, and the data are presented as the average ± SD [35 (link)].

Two-dimensional chondrogenic induction was performed as previously described [32 (link)]. Briefly, cells (1.5 x10⁵) were suspended in 5 μl of chondrogenic medium (DMEM/F12 (Life Technologies), 1% (v/v) ITS1 mix (BD), 0.17 mM AA2P (Sigma), 0.35 mM Proline (Sigma), 0.1 μM dexamethasone (WAKO), 0.15% (v/v) glucose (Sigma), 1 mM Na-pyruvate (Sigma), and 2 mM GlutaMax (Life Technologies) supplemented with 40 ng/ml PDGF-BB (R&D System) and 1% (v/v) FBS (Nichirei, Inc., Tokyo, Japan)). They were subsequently transferred to fibronectin-coated 24-well plates (Corning, Inc., NY, USA). One milliliter of the chondrogenic medium was added after 1 h. TGFb3 (R&D System) was subsequently added at 10 ng/ml on days 6 to 10. Differentiation was confirmed on day 10 using Alcian Blue staining.

Primary-cultured rat ependymal cells on fibronectin-coated 24-well plates (Corning Incorporated Life Sciences) were washed with ECF buffer, and incubated with 0.5 μCi/mL (19 nM) [³H]L-Glu at 37°C for 10 min in the absence or presence of inhibitors. After the assay, the cells were rinsed 3 times with ice-cold ECF buffer, lysed in 1 M NaOH, and neutralized with 1 M HCl. The radioactivity derived from [³H]L-Glu was measured in a liquid scintillation counter (AccuFLEX LSC-7400). A DC protein assay kit (Bio-Rad, Hercules, CA, USA) with BSA as a standard was employed to quantify the amount of cellular protein. The cellular uptake of [³H]L-Glu was evaluated as the cell/medium ratio according to Eq. 3. $\mathrm{Cell}/\mathrm{medium}\mathrm{ratio}\left(\mathrm{\mu L}/\mathrm{mg}\mathrm{protein}\right)=\frac{{}^3\mathrm{H}‐\mathrm{Radioactivity}\mathrm{per}\mathrm{cell}\mathrm{protein}\left(\mathrm{dpm}/\mathrm{mg}\mathrm{protein}\right)}{{}^3\mathrm{H}‐\mathrm{Radioactivity}\mathrm{concentration}\mathrm{in}\mathrm{medium}\left(\mathrm{dpm}/\mathrm{\mu L}\right)}$