Anti cd95 antibody

Briefly, 450 μl isolated human neutrophils (5 × 10⁶ cells/ml) were suspended in RPMI complemented with 10% FCS and 1% PEST and incubated for 30 min at 37°C in the presence of 5% CO₂. Next, 50 μl of the methacrylates (dissolved in DMSO, 500 μM), alone or in combinations (Table 1), were added to the neutrophils. For controls, buffer, proapoptotic anti‐CD95 antibody (10 μg/ml, BioLegend) or antiapoptotic LPS (Escherichia coli (serotype O127:B8) lipopolysaccharide (LPS), 100 ng/ml, Sigma) were used (Christenson, Bjorkman, Tangemo, & Bylund, 2008; Park, Jin, Song, Park, & Kwak, 2007; Ren et al., 2008) and the neutrophils were further incubated overnight (for 20 hr).
The next day the neutrophils were evaluated for cell death and the cell‐free supernatants were saved for evaluation of IL‐8 content as described below.

For apoptosis assay, HTR8 cells were digested by 0.25% trypsin without EDTA. Then, single-cell suspensions were stained with APC-Annexin V and propidium iodide in Annexin V binding buffer (BioLegend) according to the protocol. Cells were analyzed using BD FACS CantoII. FACS data were analyzed using FlowJo software (Tree Star).
To inducing the evident apoptosis of HTR8 cells, cells were planted in 24-well plates coated by 1–5 μg/mL purified anti-CD95 antibody (BioLegend) and cultured for 24 h. Then these cells were collected and detected.