Fitc anti human active caspase 3

Jurkat T cells at 1 × 10⁵ /mL were incubated with thiram at 0 (0.1% DMSO), 0.0625, 0.125, 0.25, 0.5 or 1 µM for 16 h at 37 °C in a 5% CO₂ incubator, harvested, and washed twice with PBS. The cells were fixed/permeablized with Cytofix/cytoperm solution for 20 min at 4 °C, and active caspase-3 was stained with FITC-anti human active caspase-3 for 30 min at room temperature according to the manufacturer’s instructions (BD PharMingen). Again, the flow cytometric analysis was performed with FACScan (10,000 cells per analysis) [8 (link),9 (link),10 (link),11 (link)].

Fixation/Permeabilization Solution Kit (BD Biosciences) was used to fix and permeabilize the cells according to the manufacturer’s recommendation. Briefly, 0.5 × 10⁶ neutrophils were treated with either PBS (vehicle control) or 500 ng·mL⁻¹ a2NTD for eighteen hour at 37 °C in CO₂ incubator. Cells were suspended in 250 µL of fixation/permeabilization solution for 20 min and then washed with Fix/Perm/Wash buffer. Cells were then exposed to direct or indirect staining. Direct immunofluorescence was performed using FITC anti‐human active caspase‐3 (BD Biosciences) for 30 min. The indirect immunofluorescence staining was done in two steps. First, we stain with the primary antibody as anti‐human cleaved caspase‐8 (Asp384) (Cell Signaling, Danvers, MA, USA) or anti‐human cleaved caspase‐9 (Asp315) (Cell Signaling) for 30 min followed by the secondary FITC‐conjugated antibody (Thermo Fisher) for 30 min. Isotype controls showed negative staining.