For reproducible results, each of the following protocols requires thorough knowledge of the growth characteristics of the chosen cell type. Careful control of seeding densities and cell confluence levels is essential and is best achieved by careful counting of cells and consistent feeding and treatment. We used HEK293 cells at passages 40 to 46 in the protocols that follow. Experimental hypotonic medium (200 mOsm/kg) is NaCl-free medium (Biofluids) to which NaCl is added. Hypertonic medium (500 mOsm/kg) is normotonic medium (300 mOsm/kg) to which NaCl is added. Media substituted during an experiment are equilibrated in the incubator for at least 2 h to minimize shifts of pH or temperature and plates are handled individually to minimize exposure to room air.
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