Accury c6

Murine blood samples were obtained by retroorbital puncture using citrated capillary. Whole blood samples were centrifuged at 1,300 g for 5 min and resuspended in 1 mL of Tyrode's buffer (134 mM NaCl; 20 mM HEPES; 12 mM NaHCO₃; 5 mM glucose; 2.9 mM KCl; 1 mM MgCl₂; 0.34 mM Na₂HPO₄; pH 7.4). Due to the limited volume of blood samples obtained, flow cytometry was used to test platelets activation and aggregation as previously reported (32 (link)). Aliquots of diluted blood (30 μL) were incubated for 15 min at room temperature with the appropriate anti-mouse monoclonal antibody: APC-conjugated rat anti-integrin alpha IIb (GPIIb, CD41) (6.7 μg/mL; clone MWReg30; eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) and PE-labeled rat anti-mouse integrin αIIbβ3 (GPIIbIIIa, CD41/61) (25 μg/mL; clone JON/A; Emfret Analytics, Würzburg, Germany) were used to detect platelets activation. PE-labeled CD9 and FITC-labeled CD9 (5 μg/mL; clone MZ3; BioLegend, San Diego, CA, USA) were used to visualize aggregation and the double positive population was considered the aggregated platelets. Thrombin (1 U/mL) was added when necessary. To remove excess antibody, aliquots were centrifuged (2,250 g for 5 min) and resuspended in 150 μL of Tyrode's buffer. Analysis was immediately performed in a BD Accury™ C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

DHE (150 mM) and 1 µL of the EOs of C. sinensis (0.842 µg) or C. latifolia (0.872 µg) was added to HaCaT cell (2.5 × 10⁵ in 24-well plates), which were then incubated at 37 °C for 30 min in the dark, to determine the intracellular •O²⁻ concentrations. Samples were treated with 250 µM H₂O₂ to create oxidative stress. Cells were then washed, resuspended in PBS 1X, and maintained on ice for immediate detection by flow cytometry (BD Accury C6, CA, USA). Data were analyzed using the Flowjo v10.3 software. For fluorescence quantification samples were acquired in triplicate, and 10,000 events were used for each measurement. Cells were excited at 488 nm, and DHE fluorescence was detected using 585/42 (DHE) bandpass filters. Data were expressed as the geometric mean fluorescence intensity (MFI).

PRP treated with ibrutinib, acalabrutinib, or vehicle was labeled with anti-CD42b and anti-CD62P antibody. CD42b is a membrane glycoprotein constitutively expressed on platelet surface and is useful in identifying platelet population, while CD62P is a marker of activated platelets. Labeled platelets were then incubated with swollen, heat-inactivated A. fumigatus conidia at an E:T ratio of 100:1. After 30, 60, 90, 150, 210, and 270 min, samples were acquired and analyzed on a BD Accury C6 flow cytometry. Analysis was performed gating CD42b+ cells and analyzing the mean fluorescence intensity in the positive CD62P population. Results are reported as percentages of CD62b expression normalized on DMSO-treated unstimulated platelets (100%). Normalization was performed by dividing the value of ibrutinib- or acalabrutinib-treated, stimulated or unstimulated samples to the value of the corresponding DMSO-treated unstimulated samples and multiplying by 100.

To remove contaminating iron and deplete intracellular iron storage, Ostreococcus strains were first acclimated during 5 days in 1.5 mL of AQUIL medium containing 5.4 nM Fe(III)-EDTA (1:1). To study the growth response function of iron supply, triplicate flasks containing various concentrations of Fe(III)-EDTA ranging from 5.4 nM to 270 nM Fe(III)-EDTA in Aquil medium, were inoculated with 10⁶ cells mL⁻¹ of iron-depleted cells. These cultures were grown at 20 °C, under 25 µmol quanta m⁻² s⁻¹ of continuous blue light, as described above. Cell density and chlorophyll (Chl) red fluorescence were measured using a flow cytometer (BD Accury C6). Growth rates were computed as the slope of a Ln (Nt) vs. time plot, where Nt is the cell abundance (cell/mL) number at time t. Average cell diameter was estimated using a CASY multi-channel cell counting system. (Schärfe System GmbH, Germany).

Cell growth was determined with a flow cytometer (BD Accury C6), as described previously [9 (link)].