Pr 100sa

We used artificial seawater with salinity adjusted to 37‰ for all culture steps, which was prepared by dissolving 40 g (w/v) of RedSea salt mixture (https://www.redseafish.com/red-sea-salts/) in reverse-osmosis (RO) grade water (GE Merlin). For culture of embryos and planula larvae, sea water filtered by 0.22 µm Millipore filters (MFSW) was used. So far, the only commercial brand of sea salt that we have used successfully is RedSea Salt, which is reconstituted partially from natural sea salt. When we used chemically reconstituted artificial sea water, we observed ovulation failure of jellyfish. Salinity of sea meter was checked regularly using a digital reflectometer (PR-100SA, Atago). If salinity increased to 38‰ or higher due to evaporation, RO water was added to the culture system reservoir to adjust to 37‰. Clytia can survive for short periods (a few days) in a salinity range of 35‰ to 42‰.

Standardized quantities of 0.5 mL of sputum and 0.5 mL of distilled sterile water were vortexed in an Eppendorf tube. Samples were centrifuged at 10,000x g for 10 minutes. Supernatant was then heated at 95°C for 60 minutes for microorganisms’ inactivation and protein precipitation. Following a 5-minute cooling step at room temperature, salinity was measured using a spectrophotometer lens (Digital refractometer PR-100SA, Atago, Tokyo, Japan) according to manufacturer's instructions. Two separate measurements were performed on every sample to assess same sample intermeasurements variability. Recommended quality controls and calibration using de-ionized water were performed between each measurement. Sample pH was measured using pH test strips (Sigma-Aldrich, Saint-Quentin Fallavier, France) according to manufacturer's instructions. Neutrophils intracellular pH and epithelial cells chloride channels stimulation have been shown to be altered in the context of CF.^{[16 (link),17 (link)]} Therefore, a microscopic examination and Gram staining of the supernatant was performed on each sample to confirm the absence of cellular or bacterial material which could interference with the salinity and acidity measurement.