2.8. Assessment of Apoptosis by Immunofluorescence Assay (IFA)

For the apoptosis assay, YO-PRO-1 reagent can be used as a sensitive indicator of the apoptosis [46]. Continuously increasing green fluorescence alone indicates apoptotic cells, while the simultaneous red and green fluorescence indicates dead cells. Here, we use YO-PRO^TM-1/ Propidium Iodide (PI) (Vybrant Apoptosis Assay kit 4; Thermo Fisher, Waltham, MA, USA) to assess the apoptosis of S. aureus-induced MAC-T cells. In brief, 1 × 10⁵ MAC-T cells per well was seeded in 96-well culture plates, then washed three times and infected by 1 × 10⁶ cfu/mL of S. aureus (MOI = 10) or plus with scFvs (final scFv or mixture concentration was 40 μg/mL) at a range of time points. Next, 100 μL of standard staining working solution was added to the respective well in darkness at 37 °C for 20 min. After being dyed, the stained MAC-T cells were visualized under the epi-fluorescence microscope (EVOS FL-2, Life technologies, New York, NY, USA) [47].