FIG. 2.

Impacts of selected experimental variables on TLR ligand-induced TNF. Values are means ± standard deviations for TLR responses to 0.1 mg/ml Pam3CSK4, 0.1 mg/ml zymosan, 2.5 mg/ml poly(I:C), 0.1 mg/ml LPS, 10 μg/ml flagellin, 10 mM loxoribine, and 50 μM ODN 2216. At least three repeat experiments were performed for each variable evaluated. (A) The effects of anticoagulants used for blood sample collection on TLR responses were evaluated for sodium heparin (gray bars), EDTA (black bars), and sodium citrate (white bars). Asterisks indicate values significantly different from those obtained using sodium heparin (P < 0.05). (B) The effect of serum used for cell incubation on TNF production was evaluated by incubating PBMCs with R10-FBS (gray bars), R10-HS (black bars), or RPMI containing no serum (white bars). Asterisks indicate values significantly different from those obtained using R10-FBS (P < 0.05). (C) Comparison of TNF production after 24-h (gray bars) and 48-h (black bars) incubations with TLR ligands. Asterisks indicate values significantly different from those obtained after a 24-h incubation (P < 0.05). (D) Effects of dilution and storage of ligands at −80°C (black bars) on TNF production compared to dilution of ligands at the time of the assay (gray bars). Asterisks indicate values significantly different from those obtained using ligands diluted at the time of the assay (P < 0.05). (E) Effects of PBMC isolation from blood stored at ambient temperature from 0 to 4 h (gray bars) and after 24 h (black bars) on TLR-induced TNF production. Asterisks indicate values significantly different from those obtained using blood stored 0 to 4 h (P < 0.05). (F) Differences in TNF production between PBMCs that were freshly isolated (gray bars) and those that were cryopreserved and then thawed (black bars). Asterisks indicate values significantly different from those obtained for freshly isolated PBMCs (P < 0.05).