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Page 4 of 15 Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05
[34]
cortex . In summary, some of the pharmacological activity of acetaminophen is mediated by its bioactive
metabolites p-aminophenol and AM404. It is currently unknown whether p-aminophenol and AM404
have protective effects beyond COX inhibition that may indicate the use of acetaminophen as an effective
means of treating neurodegenerative diseases. Due to resounding evidence that NO contributes directly
to the pathogenesis of AD, this study evaluated the possible beneficial effects of acetaminophen and its
metabolites in the neurodegenerative disease pathology as inhibitors of the release of NO from activated
microglia.

METHODS
BV-2 cell culture
BV-2 cells were suspended at 0.2 million cells/mL in Dulbecco’s Modified Eagle’s Medium/F12 containing
100 U/mL penicillin, 100 µg/mL streptomycin and 5% calf bovine serum (F5) (all from Fisher Scientific,
Ottawa, ON, Canada). Two mL of cell suspension were added to each well of a 24-well plate (Corning Inc.,
Corning, NY, USA) and incubated for 24 h at 37 °C in 5% CO to allow for adherence. Cells were treated
2
with various concentrations of URB597 or 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-
1-piperazinecarboxamide (BCTC) (0.02, 0.5, 2 µmol/L, Cayman Chemical Company, Ann Arbor, MI,
USA), SR144528 (0.02, 0.5, 2 µmol/L, EMD Millipore, Etobicoke, ON, Canada), indomethacin (2, 20, 50
µmol/L, Sigma-Aldrich, Oakville, ON, Canada), or vehicle solution (0.1% dimethyl sulfoxide, DMSO) and
incubated for 15 min at 37 °C in 5% CO . BV-2 cells were subsequently treated with various concentrations
2
(2, 20, 50 µmol/L) of acetaminophen (MP Biomedicals, Solon, OH, USA), p-aminophenol (Sigma-Aldrich),
AM404 (Cayman), or vehicle solution (0.1% DMSO) and incubated for a further 15 min period under the
same conditions. Subsequently, BV-2 cells were stimulated with LPS (0.5 µg/mL, Sigma-Aldrich) for 24 h to
induce NO secretion. In our experiments, the specific inhibitors URB597, BCTC, and SR144528 were used
at concentrations at least ten times higher than their reported IC50 values, thus ensuring inhibition of their
respective targets.

Griess assay for nitrite detection
Secretion of NO by murine BV-2 cells was quantified indirectly by measuring the accumulation of its stable
breakdown product, nitrite [35-38] . Briefly, 50 µL of cell culture supernatant from each well of a 24-well plate
were transferred to a 96-well plate. 50 µL sodium nitrite solutions in F5 (0.1-40 µmol/L) were also added to
the 96-well plate. An equal volume of Griess reagent, prepared immediately beforehand by combining 1%
sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% phosphoric acid (all from
Sigma-Aldrich) was then added to each well and absorbance at 570 nm was measured. Absorbance values
for test wells were normalized relative to control supernatants obtained from unstimulated BV-2 cells and
nitrite concentration was calculated from the standard curve obtained by using solutions of sodium nitrite
at different concentrations.

Lactate dehydrogenase cytotoxicity assay
Cellular death results in the loss of cell membrane integrity and an uncontrolled release of intracellular
components into the extracellular space, including the release of cytoplasmic lactate dehydrogenase
[39]
(LDH). Activity of this enzyme can be quantified to measure the extent of cell death . Briefly, 100 µL
of cell-free supernatant from each well of a 24-well plate were transferred to a 96-well plate and 20 µL
of iodonitrotetrazolium chloride (4 mg/mL, Sigma-Aldrich) were added to each well. Absorbance was
measured at 490 nm. Next, a solution was prepared consisting of lactate (750 µg/mL), b-nicotinamide
adenine dinucleotide (60 µg/mL), and diaphorase (55 µg/mL) (all from Sigma-Aldrich) in phosphate-buffered
saline (PBS); 30 µL of the solution were added to each well and absorbance was measured at 490 nm following
a 30-min incubation at 37 °C. Cell death was calculated as a percent relative to total LDH level measured in
cultures of untreated cells lysed with 1% Triton X-100 (100% lysed cells).