Fluo 8

Immortalized mouse podocytes were grown at 33 °C and cultured on 8 chamber slides (ibidi GmbH, Planegg/Martinsried, Germany). The cells were incubated with 5 μM Fluo-8 (Sigma-Aldrich, St. Louis, USA) in imaging buffer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄*7H₂O, 25 mM HEPES, 6 mM Glucose) for 30 min at 37 °C. Afterwards they were kept in imaging buffer for 20 min at room temperature. Imaging was performed with a Confocal microscope LSM710/Axiobserver Z1(Carl Zeiss Microimaging, Jena, Germany) using an 40x objective. Pictures were taken every 0.78 secs. 2 μM CNO was added after 10 pictures for the immortalized mouse podocytes and 100 μM after 20 pictures for the primary glomerular cells. 3 μM Ionomycin was used as positive control and added to the immortalized cells after 280 pictures and after 130 pictures to the primary cells.

HT-29 cells were propagated in Sarstedt culture flasks (25 cm²) in a 5% CO₂ humidified atmosphere at 37°C. Cells were fed with DMEM (Lonza) supplemented with 10% heat-inactivated FBS (Biosera) and 2 mM L-glutamine (Lonza). For the intracellular calcium measurement assay, cells were seeded in 24-well microplates (Corning CellBind Surface) at a density of 10⁵ cells/well. After 16 hr, the culture medium was replaced with fresh medium containing 5 μM of the calcium indicator Fluo-8 AM (AAT Bioquest), 4 mM of Probenecid (Sigma-Aldrich), and 0.025% (w/v) Pluronic acid F-127 (Invitrogen) according to a method adapted from Abrahamse and Rechkemmer, 2001 (link), and cells were incubated for 30 min at 37°C, 5% CO₂. The medium was removed, and calcium-free PBS was added before the cells were incubated at 37°C for another 30 min. The cell monolayer was washed twice with calcium-free PBS and PBS was added. Fluo-8 fluorescence was measured immediately after addition of Ionomycin (Sigma-Aldrich) 1 μg/ml, PBS, or purified OMVs (corresponding to 10 μg of soluble protein) with a FLUOstar Optima fluorescence plate reader (BMG Labtechnologies) fitted with custom excitation/emission filters (485/538 nm). OMVs were purified from 500 ml culture supernatants, washed with calcium-free PBS, and concentrated to a 2 ml suspension.