Z2 particle counter

Emiliania huxleyi strain AWI1516 (Alfred Wegner Institute), which produces
coccoliths, was grown in artificial seawater medium Aquil, prepared according to
the recipe of the National Center for Marine Algae and Microbiota, at
18 °C and a 12/12-h light/dark cycle. The concentration of nitrate in
the medium was 0.2 mM and of phosphate 10 μM. The standard
medium contained 10 mM CaCl₂. Cells devoid of extracellular
coccoliths were obtained by adding 1/50 volume of 0.5 M EDTA pH 8.0 to
cultures and subsequent washing with fresh medium. This treatment dissolved
extracellular calcite, but the calcite of coccoliths in statu nascendi(intracellular) remained undissolved. Cultures virtually free of coccolith
calcite were obtained by repeated cultivation of EDTA-decalcified cells in
modified artificial seawater medium, which contained 100 μM
CaCl₂. At 100 μM Ca²⁺, cells
continued to divide, whereas coccolith formation was ceased (Supplementary Fig. 1). Calcite induction
experiments were performed on calcite-free, logarithmic phase cells, 2 h
after the start of the light phase. Coccolith formation was induced by the
addition of CaCl₂ to a final concentration of 10 mM. Algal
cell density was measured using a Beckman Coulter Z2 particle counter.

Emiliania huxleyi strain AWI1516, which produces coccoliths, and its closely related sister strain CCMP2090, which does not produce coccoliths, were grown in artificial seawater medium Aquil, at 18 °C and under 50 µE light in a 12/12-h light/dark cycle. The concentration of nitrate in the medium was 0.2 mM and of phosphate 10 µM. Under standard growth conditions, where the cells form calcite, the medium contained 10 mM CaCl₂ (std-Ca medium), while under low-Ca growth conditions, where the cells do not form calcite, it contained 0.1 mM CaCl₂ (low-Ca medium). In experiments with cells from low-Ca growth conditions, the cultures had been kept in a low-Ca medium for at least 2 weeks before use. For cells inoculated into low-Ca medium, the extracellular coccoliths were dissolved with EDTA, and cells were washed with Ca-free medium before inoculation, as previously described^{20 (link)}. Algal cell density was measured using a Beckman Coulter Z2 particle counter.

Following BAL, the pulmonary and systemic circulation was rinsed with saline, supplemented with 5 mM EDTA. The left lung was used for histology, as described previously^{14 (link)}. The major lobe of the right lung was taken and thoroughly minced, enzymatically digested and subjected to red blood cell lysis. After passage through a 50 µm cell strainer, cells were counted with a Z2 particle counter (Beckman-Coulter, USA) and left on ice until labeling for further flow cytometric analysis. Another lobe of the right lung was stored for RNA extraction which was later used for miRNA profiling.

A Beckman Coulter Z2 Particle Counter was used to count the cell number. To measure the cell size, cells (n > 300) were fixed in 2% (w/v) formaldehyde buffer and bright-field microscopy images were obtained using an Olympus BX43 microscope with a DP72 camera at 200 × magnification. Cell length and width were measured by the Olympus software cellSens Dimension v.1.6. Data are presented as mean ± standard deviations.

Cell number (cells ml⁻¹) was estimated from optical density (OD) measurements (turbidity) at $750$  nm using a Thermo Scientific Evolution 201 UV–Visible spectrophotometer. OD measurements are taken at $750$  nm since this is outside the range of absorbance of pigments (phycobilins, chlorophyll a and carotenoids) in the cyanobacterium. The $\text{OD}750$ readings were converted to cell number using standard curves calibrated by the authors over the growth cycle of the cyanobacterium. The spectrophotometer specific conversion factor was $2.95·{10}^{-9}$  ml cells⁻¹ cm⁻¹ A. Samples were diluted to ensure $\text{OD}750\le 0.8$ to remain in the linear region of the standard curves, where the linear correlation between cell number and $\text{OD}750$ hold, and then modified with the dilution factor to obtain the final concentrations. A Beckman Coulter Z2 Particle Counter was used to obtain cell number for calibration of the standard curve. All readings were adjusted with the background particle counts obtained using the pure diluent. In addition, the particle counter was used to obtain mean cell volumes.

Cells were seeded into 12-well culture plates (Eppendorf) at the density of 2 × 10⁴/cm², cultivated for 24 h before the addition of dendrons and bola dendrimers. After 96 h, the cells were harvested and counted in the Z2 particle counter (Beckman-Coulter, Indianapolis, IN, USA) to calculate proliferation kinetics. For the estimation of pro-apoptotic potential of the compounds, the cells were seeded at the density of 2 × 10⁵/cm², and treated as described above for 48 h. Subsequently, AnnexinV/Propidium iodide assay was performed (FITC AnnexinV Apoptosis Detection Kit, BD Pharminogen™, San Diego, CA, USA) using ImageStreamX^® cytometer (Amnis Corp, Seattle, WA, USA) as described by Ryszawy et al. [42 (link)]. Data were analyzed with dedicated IDEAS^® 6.2 software (Amnis Corp, Seattle, WA, USA).