Cell Volume Measurement Methodology

Cell volume measurements were performed as previously described (Morabito et al., 2013 (link), 2014 , 2017 (link)). Briefly, HEK 293 Phoenix cells were transferred to round 3-cm diameter glass slides and successively placed in a perfusion chamber. Cell volume measurements were performed on cells sequentially exposed to the following: isotonic solution for 1 min and hypotonic solution (∼15% reduction of osmolality by omission of mannitol) for 2–3 min. Cell volume measurements were performed on roundish cells. For each experiment, 100–150 images/cell (1 image/1,293 s) were taken with a phase contrast microscope (Leica DMI 6000, with a HCX PL APO 63x/1.20 Lambda blue water immersion objective; Leica Microsystems). Cell diameter was measured for each image and, assuming the cell as a sphere, cell volume was calculated and expressed as V/V₀, where V and V₀ represent the volume of a cell at a given time and the average volume of the same cell in isotonic solution, respectively.