Coulter z2

Cell densities were measured using a particle count and size analyzer (Z2 Coulter, Beckman). The specific growth rate was calculated as follows: μ = (ln N₁–ln N₀) × (t₁–t₀)^–1, where μ (d^–1) is the specific growth rate, and N₀ and N₁ are the cell numbers at times t₀ and t₁, respectively. After filtering 10 mL of the culture, algal cells were collected and dehydrated, followed by spraying with gold, and placing on a scanning microscope to observe cell morphology. The scanning electron microscope (SEM; model XL-30, Phillips, Eindhoven, Netherlands) was operated at 2.0 kV to observe EPS and cell characteristics.

To avoid administration of dead cells it was important to assess the temporal viability of isolated hWJMSC. Cells were re-suspended in either phosphate buffer saline (PBS) or Dulbecco’s Modified Eagle’s Medium (DMEM) and kept at 4 °C. Cell viability was measured by trypan blue exclusion and automatic quantification (Coulter Z2, Beckman Coulter Life Sciences, USA) every 15 minutes (min) up to 180 min. Human WJMSC viability in PBS ranged between 80 and 85% up to 150 min and then halved at 165 min, while in DMEM cell viability was higher than 85% up to 180 min. Therefore, we chose DMEM as isolation and transplantation vehicle.

Platelet isolation was performed as previously described^{40 (link)}. Platelet rich plasma was obtained by centrifugation of anticoagulated (3.13% sodium citrate) whole blood at 340 g for 15 minutes. After another centrifugation step at 600 g for 10 minutes in the presence of 2 ng/mL Prostaglandin, platelets were washed and resuspended in calcium-free modified Tyrode buffer (138 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO3, 400 mmol/L Na2HPO4, 1 mmol/L MgCl2, 5 mmol/L D-glucose, and 5 mmol/L HEPES) and adjusted to the concentration required for the respective experiment. Platelet counts were obtained using a resistance particle counter (Coulter Z2, Beckman Coulter, Krefeld, Germany).

Cell proliferation under serial dilutions of CuCl₂·2H₂O was determined by quantification of the cells adhering to the culture plates after 24 and 48 h. Attached cells were trypsinized with 300 µl of an aqueous solution containing 0.25% (v/v) trypsin and 0.5 mM EDTA (Sigma, Munich, Germany). The enzymatic reaction was stopped with 700 µl of RPMI 1640 with FCS (10%) and the number of cells was measured with a cell-counter (Coulter Z2, Beckman, Krefeld, Germany).
Cell mitochondric activity as a marker for cell vitality was investigated with the WST-1 test assay (Roche, Basel, Switzerland), measuring the reduction of a tetrazolium salt to formazan. In detail, tissue culture plates with the attached fibroblasts were washed with 1 ml of Dulbecco’s phosphate buffered saline by careful rinsing to remove non-adherent cells followed by incubation for 75 min in a mixture of 750 µl RPMI 1640 with 10% FCS and 10 µl WST-1 ‘2-(4-iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-di-sulfo-phenyl)-2H-tetrazolium, monosodium salt’. After complete dissolution of the accumulated formazan in the culture medium, the amount of formazan was quantified with an UV–Vis spectrometer [λ = 430 nm, 690 nm (background), DU 640, Beckman, Krefeld, Germany].

A chronic toxicity test was conducted on P. subcapitata with 72 h exposure under static conditions to 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L of both ELT-dg and ELT-dp suspensions [47 ]. We evaluated the percentage of cell growth rate and yield inhibition at 24, 48 and 72 h. The organisms were cultured in the facility of ChemService Controlli e Ricerche s.r.l. in a climatic chamber at 24 ± 2 °C, 4440–8880 lx in the spectral range of 400–700 nm and maintained under continuous shaking to guarantee both the cell suspension and the correct concentration of carbon dioxide (CO₂). Only the algal cultures showing an exponential growth were used in the exposure, and the procedures were conducted under a laminar flow hood to avoid algal contamination. For the exposure, 100 mL capacity conical glass flasks with algal medium (see Supplementary Materials for composition) were used, with 3 replicates for each concentration and 6 replicates for the control. In each flask, 50 mL of test solution was poured and inoculated with the algae (10⁴ cells/mL). Algal density was measured every 24 h by diluting an aliquot in 9 g/L sodium chloride (NaCl) solution and reading the algal cell amount with a cell counter (Beckman coulter Z2).

To calculate the cumulative cell growth, cell counting was performed at three different time points: immediately before irradiation, 24 hours and 5 days after irradiation using an automated cell counter (Coulter Z2, Beckmann Coulter GmbH, Krefeld, Germany) (Fig 3). For this experiment, the cells were seeded into 12-well culture plates at a density of 20×10³ cells per well in a total volume of 1 mL.