Laminin coated dishes

Arl13b-mCherry-GECO1.2^tg: mIMCD cells were isolated as described³¹ . Briefly, 10 kidneys isolated from P14-P21 Arl13b-mCherry-GECO1.2^tg mice were cut longitudinally with fine scissors and the outer and inner medulla removed. The tissue was cut into small pieces with a razor blade and digested in collagenase (2 mg/ml) and hyaluronidase (1 mg/ml) for 1 h at 37°C in L-15 medium (Life Technologies). After trituration of the homogenate, cells were washed x2 in PBS and plated on laminin-coated dishes (Life Technologies). Cells were grown in DMEM (adjusted to 600 mOsm with urea and NaCl), containing 200 µM dibutyryl-cAMP (db-cAMP), unless stated otherwise. After 2 days, cells were split on laminin-coated coverslips (NeuVitro) and imaged after culturing for an additional 1–2 days to allow confluent cell growth. For side-view imaging cells were grown on 24 mm transwell inserts (corning) until they reached confluency. The membrane was excised with a scalpel and folded before imaging. Where indicated mIMCD cells where serum starved in DMEM containing 0.2% BSA for 24 h or 48 h. Imaging solutions: L-15 medium (1.3 mM Ca²⁺) or HEPES-containing solution buffered to 50 nM [Ca²⁺], see Calibration of Sensor for buffer composition.