Multisizer 4 coulter counter

HeLa cells were seeded at 10⁴ cells per well in a 24-well plate. After the measurement of cell number on day 1, hyperosmotic cellular medium (Table S2) and 5 µM of SB203850 or 10 µM of WNK463 were added where indicated. Cell proliferation was determined by counting cells at the indicated times using the Multisizer 4 Coulter Counter (Beckman-Coulter) and normalizing to day 1.

S. cerevisiae strains and wild-type S. paradoxus were grown overnight at 30^oC to OD₆₀₀ ∼0.3. Cell size and count for each sample were individually assayed: Next, the cultures were diluted 1:40 with 0.5M NaCl and immediately measured in Multisizer4 COULTER COUNTER (Beckman Coulter). A fixed amount of ODs of S. paradoxus cells was added to twice as many ODs of each S. cerevisiae sample, such that the OD ratio between them is constant throughout the samples. The mixed samples were then flash frozen.
RNA extraction and library preparation were performed as described above, and the fastq files were then processed by a pipeline for RNAseq data that was created by Gil Hornung (INCPM, Weizmann Institute of Science, Israel), as described in (Herbst et al. 2017 (link)). Total reads were normalized to the ratio between the S. cerevisiae and S. paradoxus sum-of-reads and then to the number of cells as measured in the experiment, as described earlier. Twelve repeats in SC and six repeats in Low Pi/N. Shown is the mean value with +- SE.

Cell concentration was measured using a particle sizing and counting analyser (Multisizer 4 Coulter Counter, Beckman Coulter, Brea, CA, USA). Analyses were performed for cells between 2 and 15 μm in size. A dilution of the sample was performed before analysis in order to remain within the cell concentration measurement range of the analyser. Dilution was carried out with the electrolyte of the analyser, i.e., salted Milli-Q™ water (35 g L⁻¹. NaCl). The growth rate (μ, in day⁻¹) was calculated according to Andersen [107 ], between dilutions in semi-continuous growing conditions and until the end of the expontential growth phase for the batch growing conditions.

Cell sizes were measured using a Multisizer 4 Coulter Counter (Beckman Coulter) using a spherical approximation for the P. antarctica cells. Samples were measured directly after addition to an isohaline solution, and these data were used to analyze cell size and single cell counts. Glutaraldehyde (Sigma Aldrich) was observed by visual microscopy to break up the colonies at a final concentration of 2.2% (v/v). It was added to samples that were shaken vigorously for 30 seconds and measured directly. These data were used to obtain the total (single and colonial) cell counts. The data were analyzed using the Beckman Coulter Multisizer v4.01 software. Diameter, surface area, and cell volume were analyzed separately to account for deviations from the normal distribution. As reported previously for other species [20 (link)], Glutaraldehyde changed cell size inconsistently among clones and treatments, and thus the sizes of colonial cells are not reported.

The Sf9 and HzAM1 cell lines cultured in SF900III SFM (Gibco®, Life technologies, Carlsbad, CA, USA) were incubated at 28 °C in 250 mL flasks shaking at 120 rpm on an orbital shaker. BV stocks of r-β-gal AcMNPV (a recombinant AcMNPV virus expressing the B-Galactosidase gene) [28 (link)] and HearNPV were prepared as described in Matindoost et al. (2012) [29 (link)]. Cell number and size distribution were determined using a Multisizer™ 4 Coulter Counter^® (Beckman Coulter, Brea, CA, USA).