Total production of IL-8 by neutrophils in response to various TLR ligands. (A-C) Control and mobilized neutrophils were cultured overnight with various TLR ligands. Afterward, the supernatants were collected, and the pellets were incubated in Triton-100–based buffer for 30 minutes on ice. After lysis, samples were spun down at 12 000g for 15 minutes, and the supernatants, representing the cell-associated fraction, were collected. Secreted and cell-associated levels of chemokine were measured by enzyme-linked immunoabsorbent assay. Results represent data from 6 independent experiments, with cells from 6 different donors. Error bars represent SEM for the total production of IL-8 (sum of secreted and cell-associated protein). Data were analyzed by independent 2-way analysis of variance test; *P < .05 (significant difference).