Fig. 1.

Comparison between effects of different commercial- and pharmaceutical-grade preparations in the absence or presence of PMX on a number of microglial activation parameters in the N9 cell line. N9 cells were either unstimulated or stimulated with 100 EU/mL of control standard endotoxin (CSE), 100-U/mL recombinant human α-thrombin (rh-thr 100), 10-U/mL recombinant mouse interferon-γ (rm-IFNγ), 100-U/mL plasma-derived bovine α-thrombin (pb-thr), 100-nM serum-derived human α₂-macroglobulin from supplier #5 (sh-α₂M-5), 100-nM serum-derived human α₂-macroglobulin from supplier #7 (sh-α₂M-7), 1-μM 2,4,6-trinitrophenol-conjugated egg-white-derived chicken ovalbumin (tnp-Oval), 15.4-μM (1 mg/mL) serum-derived rat serum albumin-essentially fatty acid free (sr-Alb-efaf), and/or 15.4-μM (1 mg/mL) serum-derived rat albumin-fraction V powder (sr-Alb-FV) as indicated in the absence or presence of PMX (10 μg/mL). Cell surface expression of CD95 (Fas) (A) and CD40 (B) was assessed 24 h later by flow cytometry. Values for the treatment groups that did and did not include PMX were normalized to their respective controls. Means ± S.E.M. for these values from at least three separate experiments are presented. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding preparation treatment without PMX. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. unstimulated control or PMX alone control (dotted line). Prenormalization mean absolute fluorescent intensity units ± S.E.M. for unstimulated cells were 15.9 ± 2.5 and 15.9 ± 2.8 in the absence or presence of PMX, respectively, for CD95(Fas) and 7.4 ± 1.2 and 5.5 ± 1.1 for CD40. Metabolic activity/proliferation (C), NO production (D), and TNF-α release (E) were also assessed 24 h following stimulation using the WST-1 assay, Griess reagent and cytokine ELISA, respectively. Values for the treatment groups that did and did not include PMX were normalized to their respective controls. Data are expressed as mean ± S.E.M. n ≥ 9, exp ≥ 3. Prenormalization mean absolute values for unstimulated cells were 0.32 ± 0.015 and 0.34 ± 0.032 absorbance units (C), 0.0 and 0.0 μM NO (D), and 0.0 and 1.33 ± 1.33 pg TNF-α/μg total protein (E) in the absence or presence of PMX, respectively.