Three-dimensional culture of MDCK cells on Matrigel.

3D cell cultures were performed as previously described (13). Briefly, four-well glass slides (Lab-TEK II) were coated with 85 μl of Matrigel solution (Corning) at 4°C. Matrigel was allowed to solidify for 20 min at 37°C. MDCK cells expressing EGFP-tagged NKCC1-WT, NKCC1-DFX, NKCC1–241, NKCC1–242, and NKCC1–244 mutants were resuspended in complete DMEM at 1 × 10⁴ cells/ml. A volume of 20 μl of cell suspension was added to 200 μl of 2% Matrigel. This cell suspension was then layered on top of the solidified Matrigel and incubated in a humidified incubator at 37°C, 5% CO₂. Cells were grown from 6 h to 8 days. Conditioned medium was aspirated every 48 h and replaced with fresh 2% Matrigel in complete DMEM medium. Transparent cysts with hollow lumen were visible under brightfield microscopy by day 4.