H2dcfda

The generation of ROS was determined using the H₂DCFDA-based assay, according to the methods published previously [32 (link), 33 (link)]. Briefly, H1299 cells were plated in 96-well plates at 2.5 ×10⁴ cells/well and cultured for 24 h. After twice washing with PBS, cells were incubated with 20 μM H₂DCFDA (Cayman Chemical) at 37°C for 45 min and then treated with BC-23, radiation, or a combination of BC-23 plus radiation. The fluorescence intensity (excitation/emission = 485/535 nm) was recorded for 2 h using a Synergy H1 microplate reader (BioTek).

Staining protocols adapted from Feugang et al. [37 (link)] and Martinez-Alborcia et al. were used [38 (link)]. Nanoselected spermatozoa were diluted to 30 × 10⁶ cells/mL with a pre-warmed phosphate-buffered saline solution (PBS). Aliquots of 0.1 mL of sperm suspensions were allocated to various staining for viability assessments: 2 μL propidium iodide (PI, 1 mg/mL in PBS) for plasma membrane integrity, 5 μL PNA-FITC (100 mg/mL in PBS) for acrosomal reaction, 2 μL JC-1 (500 mg/mL; Cayman Chemical Co.; Ann Arbor, MI, USA) for mitochondrial integrity, and 2.5 μL H₂-DCFDA (1 mmol/L in DMSO) for reactive oxygen species (ROS) accumulation within the cells. All samples were incubated at 37 °C for 15 min, then diluted six times with prewarmed-PBS to reduce both dye and sperm concentrations for adequate and immediate analyses with flow cytometry (Becton–Dickinson FACSDiva version 6.1.3). A total of 10,000 events were set to evaluate the proportions of stained spermatozoa (control and nanoselected) and their relative fluorescence intensities to assess sperm damage. Sample aliquots were mounted onto individual microscope slides to confirm the successful staining under an epifluorescence microscope (EVOS FL-Auto, Thermo Fisher Scientific; Hampton, NH, USA).

The starting materials for the synthesis of the loxapine-focused compound library were purchased from Sigma-Aldrich, Acros Organics, or Tokyo Chemical Industry (TCI). The synthetic details of the loxapine derivatives will be reported elsewhere. The purity of all tested compounds was determined to be >95% by proton nuclear magnetic resonance (¹H NMR) spectra with Bruker DPX400 (400 MHz) instruments. loxapine (Sigma-Aldrich), PMA (LC laboratories), H₂DCFDA (Cayman Chemical), rifampicin (Bio Basic), SW14, and other loxapine derivatives were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Hoechst 33342 (Thermo Fisher Scientific), gentamicin (Bio Basic), ampicillin (United States Biochemical [USB]), ciprofloxacin (Sigma-Aldrich), streptomycin (Bio Basic), ofloxacin (Bio Basic), and vancomycin (Goldbio) were dissolved in deionized water and filtered through a 0.45-μm filter. Tetracycline (Bio Basic) and chloramphenicol (Amresco) were dissolved in 95% ethanol as stock solutions.