Figure 7.
Necroptosis attenuates LPS-induced inflammation in vivo. (a) Female balb/c mice (4 per group) were injected (intraperitoneally, 200 μl) with PBS or zVAD (250 μg per mouse). After 1 h, mice were injected again with either PBS or LPS (10 μg per mouse). After a further 24 h, mice were killed, and the peritoneal cavity was washed with 4 ml PBS. Total cellularity was scored by microscopy. (b) Spleens were removed, and splenocytes were collected through a strainer. Erythrocytes were removed using RBC lysis buffer and then total cellularity was analysed by microscopy. (c) Peritoneal cell death was analysed by Aqua positivity. (d) Splenocytes were immunostained and analysed by flow cytometry. Population numbers were then generated by multiplying cell percentages by total splenic cellularity counts. Neutrophils (CD11b+, Gr1hi, F4/80med, SiglecF-), eosinophils (SiglecF+), B-cells (CD19+), T-cells (CD3+). (e) Peritoneal-derived cells from control mice were seeded at 1 × 106/ml in 24-well plates. Cells were pretreated with zVAD (20 μM) or not, followed by addition of LPS to 10, 5 and 1 μg/ml. After 48 h, cell death was analysed by forward scatter/PI staining, and cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. Error bars represent the mean±S.E.M. *P<0.1; **P<0.05; ***P<0.01 by Student's t-test