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Davis et al. Neuroimmunol Neuroinflammation 2018;5:50 I http://dx.doi.org/10.20517/2347-8659.2018.60 Page 3 of 11

in vitro; and to a large extent this is because most in vitro studies utilize rodent cells and these are the stimuli
to which they respond. Therefore, the primary objective of our investigation was to advance our knowledge
of IL-1β-induced inflammatory signaling in human microglia using C20 human microglial cells.

METHODS
Cells
C20 human microglial cells were obtained from David Alvarez-Carbonell, PhD (Case Western Reserve
[37]
University) and details pertaining to the generation of this cell line were recently reported . Briefly, these
investigators obtained human microglia from ScienCell Research Laboratories, Carlsbad, CA (Cat# HM1900)
and then immortalized the cells using simian virus 40 large T antigen and hTERT (to facilitate expression of
[37]
human telomerase reverse transcriptase) . The C20 cells that we obtained were confirmed to be of human
origin by the Human Identity Testing Laboratory at Oklahoma State University Center for Health Sciences,
which utilized the PowerPlex® 21 System (Promega, Madison, WI), a multiplex short tandem repeat system
[44]
for human identification, as previously described . For our experiments, cells were used at passages 5-10
6
5
and were either seeded in 24-well plates (1 × 10 cells/well) or in 100 mm dishes (3 × 10 cells) depending
on the experiment and cultured 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% fetal bovine serum (Atlanta
Biologicals S11550), and 1% penicillin/streptomycin (Lonza 17603E)] until 90% confluent (4-5 days). Medium
was replaced with serum-free medium (SFM) 24 h prior to stimulation. Normal human astrocytes (NHA,
[45]
ScienCell, #HA1800) were maintained as previously described .
Stimulus
C20 were stimulated in SFM containing either LPS (E. coli K12, 1 µg/mL; InvivoGen, San Diego, CA) or
human recombinant IL-1β (20 ng/mL; Peprotech, Rocky Hill, NJ) for 10 min - 24 h depending on the specific
experiment; whereas, NHA were stimulated with IL-1β (3 ng/mL) for 24 h in the single study in which they
were used. Details regarding the number of independent experiments and replicate treatments within each
experimental run are provided in the figure legends.

Expression of microglial marker
While the precise function of transmembrane protein 119 (TMEM119) has yet to be determined, it is
increasingly recognized as a reliable marker of human microglia that discriminates microglia in the
brain from blood-derived macrophages [46-49] . We assessed TMEM119 expression by western blot analysis
and fluorescent immunocytochemistry. For western blot analysis, whole cell lysates were collected from
unstimulated and IL-1β-stimulated C20 cells (cultured in 100 mm dishes), using Triton X-100 lysis buffer
(50 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100) containing MS-SAFE protease/
phosphatase inhibitor (Sigma-Aldrich). Briefly, cells were rinsed with cold phosphate buffered saline (PBS),
then lysed in 300 µL of lysis buffer and collected into 1.5 mL tubes. The lysates were then incubated on ice
for 45 min with intermittent mixing by inversion. Lysates were centrifuged for 10 min at 20,800 × g and 4 °C.
The supernatant, containing whole cell protein was then collected and stored at -80 °C. Thirty micrograms
of total protein were loaded on 7.5% polyacrylamide gels (BioRad TGX FastCast Acrylamide kit, #161-0171),
electrophoresed and then transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were
then incubated at 4 °C for 15-18 h with rocking. Membranes were blocked with 5% bovine serum albumin
(BSA) for 2 h prior to incubation with antibodies. Primary antibodies included, anti-TMEM119 (1:100, Sigma
#HPA051870), anti-glial fibrillary acidic protein (GFAP; 1:5000, Millipore #MAB360), and anti-β-tubulin
(1:1000, Cell Signaling #2146S). Anti-rabbit IgG, AP-linked (1:1000, Cell Signaling #7054S) was used as the
secondary antibody. Whole cell lysates from NHA were used for comparison. Restore western blot stripping
buffer (Thermo Scientific #21059) was used to remove antibodies and allow for re-labelling of membranes. The
blots were scanned in a phosphoimager Typhoon 9410 (GE Healthcare, Uppsala, Sweden) using Amersham
ECF Substrate #RPN-5785, and Image J (National Institutes of Health) was used for densitometric analysis.