Rhodamine 123 r123

Fluo-3AM, FluoZin-1, FluoZin-3AM, 6-Carboxy-2′,7′-dichlorofluorescein diacetate (DCF), and rhodamine 123 (R123) were obtained from Molecular Probes (Eugene, OR, USA). ⁴⁵CaCl₂ was produced by Polatom Sp. z o.o., Otwock—Swierk, Poland. Other chemicals, including nanosilver and cell culture materials were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All reagents were of analytical grade.

The percentage of live sperm cells with functional mitochondria was assessed using a combination of fluorescent stains Rhodamine 123 (R123) (Molecular Probes, OR, Eugene, USA) and propidium iodide (PI) as described previously. In each slide, 200 spermatozoa were examined under an epifluorescence phase-contrast microscope (Eclipse Ts 2, Nikon, Tokyo, Japan) at 600× magnification, equipped with an excitation/barrier filter of 490/515 nm for R123 (blue excitation), excitation/barrier filter of 545/590 nm for PI (green excitation), and a digital camera (Olympus DP 11, Tokyo, Japan). The sperm cells displaying green fluorescence in the mid-piece region and no red fluorescence in the head were considered viable with functional mitochondria, whereas cells exhibiting red fluorescence in the head were counted as dead [54 (link)].

To assess mitochondrial impairment induced by v- and ox-nPS/mPS, we measured transmembrane potential by the incorporation of the fluorescent probe rhodamine 123 (R123) (Invitrogen Molecular Probes, Eugene, OR, USA). The chemical properties of the cationic fluorochrome R123 allow mitochondrial membrane crossing and storage in the matrix only in functional mitochondria that possess a transmembrane potential (ΔΨm), which is indicative of an active proton gradient maintained during oxidative phosphorylation [41 ]. A549 monolayers, grown in 96-well plates, were treated with 100 μg mL⁻¹ of v- and ox-nPS/mPS suspensions for 24 h. After incubation at 37 °C and washing in PBS to remove the non-internalised particles, cells were treated with the probe solution (10 μM final concentration) and incubated for 10 min at 37 °C. Fluorimetric readings were carried out using a microplate reader (Tecan Italia, Milan, Italy) set to 535 and 595 nm as the excitation and emission wavelengths, respectively. In comparison to the control cells, the percentage changes of emission values were calculated for each sample.