Pi staining kit

All chemicals [AgNO₃ (209139), PBS (P3813), DMSO (C6164), tryptic soy broth (B311-150-S), Sabouraud-gentamicin-chloramphenicol (89579), RPMI agar (R8758), and phenazine methosulfate (P9625)] employed in this study were acquired from Merck (Darmstadt, Germany) and Sigma Chemicals Co (St. Louis, MS, United States). Mueller-Hinton broth (CM0405B), CellROX™ Green (C10444), Hoechst 33342 and PI staining kit (V13244) were purchased from Thermo Scientific™. Dulbecco’s Modified Eagle’s Medium (51435C), cell proliferation kit I (MTT) (11465007001), and cell proliferation kit II (XTT) (11465015001) were acquired from Merck (Darmstadt, Germany) and Sigma Chemicals Co (St. Louis, MS, United States). For the comparative biological study, commercially available chemically synthesized Ag NAPs (795925) were purchased from Sigma-Aldrich and denoted as CHE-Ag NAPs.

Cell samples were fixed in 10% formalin washed in Hank's solution, and blocked using PBS supplemented with 10% goat normal serum. Samples were then washed with PBST and then treated with primary antibodies at 4°C overnight. Subsequently, samples were then incubated with fluorescence-conjugated secondary antibodies. After stained with DAPI, areas of interest were captured by a Zeiss Axio Observer Z1 microscope.
Apoptosis was assessed by propidium iodide (PI) staining using a PI staining Kit (P1304MP, ThermoFisher). Images were acquired using a Zeiss Axio Observer Z1 microscope.