Multisizer 2

To determine culture viability, small aliquots of culture broth were taken from the reactor and cells were counted on a Z2 Coulter Counter (Beckman Coulter, Woerden, Netherlands) equipped with a 50 µm orifice (Multisizer II, Beckman Coulter, Woerden, Netherlands). Cells were diluted in 0.1% peptone and 100 µL suspensions containing approximately 30, 300 and 3000 cells were plated on yeast extract (1% w/v), peptone (2% w/v) and dextrose (0.5% w/v) (YPD) agar plates and incubated at 30°C for at least 3 days before counting the colonies. CFU was calculated as the number of colonies formed divided by the number of plated cells.

Cells were harvested using trypsin/EDTA and reseeded into 48-well plates at a density of 20,000 cells/well in fresh media containing treatments as indicated. Cells were allowed to grow for 10 days with medium changes every 3 days. At the end of the experiment, the medium was removed and 0.5 ml of trypsin/EDTA was added to each well. Once the cells were in suspension, cells were drawn into a 5 ml syringe through a 25G needle three times to obtain a single-cell suspension. The wells were then washed with 0.5 ml of fresh Isoton II solution and this was then drawn up into the syringe. This final wash was repeated twice to give a total volume of 2 mls in the syringe. The solution was then added to 8 mls of Isoton II solution in a counting vial to make up a volume of 10mls. Cells were then counted (three counts per well) using a Coulter™ Multisizer II.

Fat from all Block 1 males was used in this study. The reserved 50–60 mg portion of the fat pad (above) was prepared for cell counting using a modification of the method of Hirsch and Gallian [53 (link)] as described by Kump and Booth [54 (link)], and cell number and size distribution were measured by the Coulter method [55 (link)]. Briefly, the fat was fixed and adipocytes were separated from other tissue in the OsO₄-collidine solution for 3–4 weeks; this procedure results in free cells. The cells were washed with isotonic saline solution and left in saline for 24 h, and then washed with 8 M urea in saline and left in that solution for 3–4 days. The cells were finally rinsed with 0.1% Triton X-100 and filtered through a 250 µm filter onto a 10 µm filter. The collected cells were suspended in Isoton II (Beckman-Coulter, Fullerton, CA, USA) containing 10% glycerol. Cells were counted and cell size was determined on a Coulter Multi-Sizer II, with the particle counting window set to 8.03–271.1 µm. Cells were counted in three 15-s bursts, and the three counts were summed to give a single measure per 45 s period. Cell counts reported here are the total number of cells contained in the combined fat pads.

RCC299 and CCMP1545 were grown under a 14 h/10 h light/dark cycle in L1 media in artificial seawater (as above) at 220 μE m⁻² s⁻¹ PAR. Both strains were maintained in light-acclimated, mid-exponential growth before experiment initiation. Two days before the experiment start cultures of each species were split into duplicates A and B. Ten mM Penicillin G (final concentration, i.e., 6000 Units ml⁻¹) and 10 mM Fosfomycin (Sigma-Aldrich) were added 1 h after lights on (at T₀). At each time point cells were fixed in 0.25 % glutaraldehyde (final concentration) for 30 min in the dark and frozen in liquid N₂. Cells were measured using an Influx flow cytometer (BD Biosciences) and analyzed using Winlist (version 7.1, Verity Software House). Forward angle light scatter (FALS) and SSC were normalized using 0.75 μm diameter YG beads (Polysciences Inc.) and chlorophyll fluorescence (692 ± 40 nm band pass) was normalized to 2 μm diameter Polychromatic Red beads (Polysciences, Inc.).
To measure RCC299 for the morphological description, >10,000 cells from a mid-exponential phase, axenic culture were measured live on a Coulter Multisizer II approximately midway through the light period. Cells were grown on a 14 h/10 h light/dark cycle in K medium in artificial seawater maintained in mid-exponential growth for >10 generations after acclimatization to 21 °C and 90 μmol photons m² sec⁻¹ PAR.

Lung BAL cells were counted using a Coulter Multisizer II (Coulter Canada, Ville St-Laurent, QC, Canada), and differential cell counts were obtained from cytospin preparations using Wright stain and following standard procedures [48 (link)].

Growth assays were performed as reported previously [9 (link)]. Briefly, the growth rates of three cell lines were compared as follows: MCF-7, TamR and FasR cells were grown to 70% confluency, passaged and seeded into 24 well plates at a density of 1.5 million cells per plate. Cells in each well were trypsinised and counted using a Coulter Multisizer II on days 1, 4, 7 and 9. The test substances PD98050, LY294002, fish oil and 4-hydroxytamoxifen (MCF-7 cells only) are detailed in Table 1. To determine the effects of the on cell growth, cells were again seeded into 24-well plates and left overnight to attach. After 24 h equilibration, base counts were taken and then cells were treated with the appropriate agents with cell counts being taken on days 1, 2, 4, 7 and 9 as above; cell growth data is presented graphically following normalisation to control. Cells were re-treated on day 4.