Lipopolysaccharide from escherichia coli o111 b4

We studied intracellular production of pro- and anti-inflammatory cytokines by B and T lymphocytes as previously described.¹⁶ In addition, we explored intracellular cytokine production by monocytes by stimulating aliquots of 10⁶ PBMCs with 1 mg/mL lipopolysaccharide (from Escherichia coli O111: B4; Merck, Kenilworth, NJ) during 4 hours at 37°C in 5% CO₂.

Cytokine release in response to isolated platelet-derived EVs was measured in whole blood using commercial ELISA kits. Whole blood from five healthy donors was diluted 1:5 in RPMI medium (Gibco, Life Technologies) and stimulated with 50 μL isolated EVs from platelets stimulated with HEPES, thrombin, or M1 protein for 24 h in a CO₂ incubator (37°C, 5% CO₂, >95% relative humidity). Heparin (0.6 μg/mL, Sigma-Aldrich) was added in combination with penicillin (100 mg/mL, Gibco, Life Technologies) and streptomycin (100 mg/mL, Gibco, Life Technologies) to prevent clot formation and contamination. Lipopolysaccharide from Escherichia coli O111:B4 (1 μg/mL, EMD Millipore Corp.) was used as a positive control for monocyte activation, and HEPES buffer alone was used to determine the background cytokine release. The cytokine release mediated by M1 protein alone (2.5 μg/mL) was also investigated. After incubation, the samples were centrifuged at 500 g for 5 min, and cytokine and chemokine levels in the supernatants were measured using commercial ELISA kits for IL-6 (Invitrogen, Thermo Fisher Scientific), human CCL2/MCP-1 (R and D Systems), and human CXCL8/IL-8 (R and D Systems), according to manufacturer instructions.