Software analysis

Sample cell size for ZnCl₂-treated M. aeruginosa UTEX LB 2385 cultures was measured (FCM) in 10,000 events per sample within a calibrated MACSQuant Analyzer 10 (Miltenyi Biotec, Auburn, CA, USA) containing 405 _nm, 488 _nm, and 638 _nm lasers, using the forward scatter setting (FSC-A) for a set short term and long term course period of 8 days (0, 0.25, 0.5, 1, 5, 8). For each time point, 1 mL of each cell treatment was aseptically transferred into a 1.5 mL microcentrifuge tubes, and the parameters were set to gently resuspend before 100 µL was measured in FCM. Different cyanobacteria populations may exhibit distinct patterns of size, complexity, and autofluorescence in regards to colony formation and to their phycobilisome complex. Furthermore, the light harvesting components and phycobilisome complex has been shown to be affected by heavy metals in concentration dependent kinetics [71 ,72 ]. Allophycocyanin and phycoerythrin fluorescent intensities for M. aeruginosa UTEX LB 2385 cells were measured in a calibrated MACSQuant Analyzer 10 (Miltenyi Biotec, Auburn, CA, USA) for the set time course period of 8 days (data not shown). All flow cytometry histograms and statistical analysis of measurements were generated via FlowJo software analysis (FlowJo, Ashland, OR, USA) to compare the effects of varying concentrations of ZnCl₂ treatment within all cultures.

For synchronization of Giardia, aphidicolin (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 5 μg/ml was added to cells grown to about 60% confluency. Control cells were treated with 0.05% DMSO which was used to solubilize 5 µg/ml aphidicolin. After a 6-h incubation at 37 °C, the medium was replaced with drug-free fresh TYI-S-33 culture medium and incubated for an additional 3 h.
Flow cytometry analysis of various cells (DMSO-treated control, aphidicolin-treated and aphidicolin-washed trophozoites) was performed as previously described [3 (link)]. Briefly, the harvested cells were re-suspended in 50 μl of TYI-S-33 culture medium and were treated with 150 μl of cell fixative (1% Triton X-100, 40 mM citric acid, 20 mM dibasic sodium phosphate and 200 mM sucrose, pH 3.0) at room temperature for 5 min. The samples were diluted with 350 μl of diluent buffer [125 mM MgCl₂ in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na₂HPO₄ and 2 mM KH₂PO₄, pH 7.4)] and then stored at 4 °C until use. Fixed cells were reacted with 2.5 μg of RNase A (Sigma-Aldrich) and 10 μg/ml of propidium iodide (Sigma-Aldrich) for 30 min at 37 °C. The cells were evaluated with respect to their DNA content by flow cytometry and FlowJO software analysis (FlowJo Llc, Ashland, OR, USA).