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Page 302 Davis et al. Neuroimmunol Neuroinflammation 2020;7:300-10 I http://dx.doi.org/10.20517/2347-8659.2020.19

METHODS
Cells
Normal human astrocytes (NHA, cat# HA1800; ScienCell Research Laboratories, Carlsbad, CA, USA)
[28]
were cultured as previously reported . Briefly, cultures were maintained in growth medium containing
Astrocyte Medium (cat# 1801), 1% Astrocyte Growth Supplement (cat# 1852), 1% penicillin/streptomycin
solution (cat# 0503), and 2% fetal bovine serum (FBS). Culture medium was replaced every 48 h.
4
Experimental cultures were seeded in either 24-well plates (1.15 × 10 cells/well) or 100-mm dishes (2.5 ×
5
10 cells/dish) and grown until 90% confluent (15 days).
C20 human microglial cells were obtained from David Alvarez-Carbonell, PhD (Case Western Reserve
[30]
University), and the details regarding the generation of this cell line have been reported . The C20 cells
that we received were evaluated by the Human Identity Testing Laboratory at Oklahoma State University
[31]
Center for Health Sciences and confirmed to be of human origin as previously described (Davis et al. ,
2018). Cultures were maintained in growth medium [Dulbecco’s Modified Eagle Medium/Ham’s F-12 50/50
mix supplemented with 2.5 mmol/L L-glutamine (Corning 10-090-CV), 10% FBS (Atlanta Biologicals
[31]
S11550), and 1% penicillin/streptomycin (Lonza 17603E)] as previously reported .

Treatment of cells
To determine the dose-dependent effect of chronic (72 h) β-FNA (NIDA reagent supply program; Bethesda,
MD, USA) on CXCL10 expression in NHA, cells were initially cultured in growth medium containing
0.04-10 µmol/L β-FNA for 24 h. The medium was then replaced with serum-free medium (SFM) containing
β-FNA for an additional 48 h; IL-1β (3 ng/mL; Peprotech, Rocky Hill, NJ, USA) was added to cultures for
the final 24 h of the 72 h exposure period. To determine the differential effects of β-FNA on chemokine/
cytokine expression, NHA were treated as described above; however, only a single concentration of β-FNA
was used (3 µmol/L; EC for inhibition of CXCL10 expression). To assess the effects of β-FNA on IL-1β-
50
induced activation of (NF)-κB p65, cells were chronically exposed to β-FNA (3 µmol/L) as described above,
then stimulated for 30 min with IL-1β (3 ng/mL). IL-1β-induced NF-κB p65 activation was assessed at 30
min after stimulation (when peak activation is observed).

This was our initial investigation into the effects of β-FNA on chemokine/cytokine expression in C20
microglial cells. Thus, we used our acute exposure model. Consistent with the acute model previously used
with astrocytes, we used a higher concentration range of β-FNA (3-30 µmol/L). Briefly, C20 microglial cells
were serum deprived for 24 h and then treated with β-FNA (3-30 µmol/L) alone or in combination with IL-1β
(20 ng/mL) for 24 h in SFM.

Cell viability
Cell viability was measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
[27]
assay as previously reported . Briefly, cells were exposed to MTT (0.55 mg/mL) for 45 min; next, the
medium was removed, and cells were dissolved in 1 mL DMSO. The absorbance was then measured at
492 nm using a BIO-TEK HT spectrophotometer.

Chemokine/cytokine expression
Standard dual-antibody solid-phase immunoassays (ELISA Development Kit, Peprotech) were used
[27]
for quantitation of cytokines/chemokines in culture medium as previously described . Values were
normalized to total protein content, which was determined using the bicinchoninic acid protein assay as
[32]
previously described .
NF-κB activation
Phosphorylated NF-κB p65 in nuclear fractions was measured as an indicator of NF-κB p65 activation.
Following experimental treatments, NHA (in 100-mm dishes) were washed two times with PBS; nuclear