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Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05 Page 5 of 15

MTT cell viability assay
MTT is a water-soluble tetrozolium dye that is converted by the mitochondrial enzyme succinate
dehydrogenase to an insoluble purple formazan in live cells. Spectrophotometric quantification of
[40]
formazan dye present in the cells allows for determination of cell viability . The MTT assay is more
sensitive in detecting cytotoxicity when compared with the LDH assay; however, it may also incorrectly
report mitochondrial dysfunction as cell death [41,42] . Therefore, the MTT and the LDH assays were used in
parallel in this study. The MTT cell viability assay was conducted by, first, adding MTT (Sigma-Aldrich)
to the cell cultures in a 24-well plate to reach a final concentration of 500 µg/mL. Plates were incubated
for 1 h at 37 °C in 5% CO to allow for cellular metabolism of the tetrozolium dye. Then 20% sodium
2
dodecyl sulfate/50% N,N-dimethylformamide (both from Fisher Scientific) solution in milliQ water
(EMD Millipore) was added 1:1 to the cell culture in each well to lyse the cells and solubilize the formazan
crystals. Plates were incubated overnight at 37 °C until the crystals dissolved and absorbance of each
sample was measured at 570 nm. Cellular viability was calculated as a percent relative to fully viable cells
incubated in fresh cell culture media only.

Statistical analysis
Data were analyzed with Prism V7.0c (Graphpad Software, Inc., USA). One-way and two-way analyses of
variance (ANOVA) were performed, followed by Dunnett’s post hoc test and Tukey’s honestly-significant
difference (HSD) test, respectively. Data are presented as means ± standard error of the mean (SEM).
P-values less than 0.05 were considered statistically significant.

RESULTS
The aim of this study was to determine whether the inhibitory effects of acetaminophen on microglia-
driven inflammation are mediated by the parent acetaminophen molecule or its metabolites, p-aminophenol
[36]
and AM404. BV-2 microglial cells were used, which is a widely-accepted model of primary microglia .
Their activation was induced by LPS, which, similar to several endogenous pro-inflammatory
molecules, triggers expression of inducible nitric oxide synthase (iNOS), leading to release of neurotoxic
concentrations of NO by microglia [43-46] .

Clinically, acetaminophen is indicated for treatment of fever and pain. It has been reported to affect several
microglial functions in vitro. In LPS-stimulated microglia, acetaminophen suppressed the synthesis of the
inflammatory mediators PGE2, PGF2, and thromboxane B2 by inhibiting the activity of COX; however,
it had no effect on the levels of the pro-inflammatory mediators TNF-α and NO [19,20] . Data in Figure 2A
confirm these observations by showing that at therapeutic as well as supratherapeutic concentrations
acetaminophen had no significant effect on NO released by LPS-stimulated BV-2 microglia. Both
metabolites of acetaminophen studied, p-aminophenol and AM404, at 20 and 50 µmol/L significantly
inhibited this parameter of microglial activation [Figure 2B and C]. It is important to note that at the
concentrations studied none of the three compounds reduced the viability of BV-2 cells, relative to vehicle-
treated control cells, measured by the LDH [Figure 3] and MTT assays [Figure 4]. Stimulation of BV-2 cells
by LPS causes significant increase in cell death (30%-40% cell death, Figure 3) and reduction in cell viability
(50%-80% cell viability, Figure 4) in the absence of any of the compounds studied. Adding acetaminophen,
p-aminophenol or AM404 did not lead to further enhancement of the LPS-induced cell death. Because
nontoxic concentrations of inhibitors were used, it was concluded that the observed decreases in NO
secretion by stimulated BV-2 microglia were caused by inhibition of specific signaling pathways.

Previous reports have suggested that at least some of the clinical effects of acetaminophen could be due
[47]
to its metabolism to AM404 or the deacetylated intermediate, p-aminophenol . Following distribution
to the CNS, p-aminophenol is conjugated to arachidonic acid through the catalytic activity of FAAH,