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Figueroa-Hall et al. TLR4-mediated signaling in CHME-5 cells

Protein extraction 5% BSA was used for all other antibodies. Primary
Depending on the experiment, either whole cell lysate antibodies diluted in blocking buffer (1:500-1:2,000),
or cytoplasmic/nuclear extractions were isolated for were added to membranes and rocked overnight at 4 °C.
subsequent assessment of protein expression. For Membranes were then washed 3 times with TBST
whole cell lysates, cells were washed with ice-cold for 5 min with each wash. Alkaline phosphatase-
phosphate-buffered saline (PBS) and then 400 μL of linked secondary antibodies diluted in blocking buffer
cell Lysis Buffer (CBL, Cell Signaling) was added to (1:1,000-1:5,000) were added to membranes and
each 100-mm dish. Following a 5-min incubation at 4 °C, rocked for 2 h at 25 °C and then washed 3 times with
lysates were collected and centrifuged at 14,000 × g TBST for 20 min. Enhanced Chemifluorescence (ECF)
(4 °C) for 10 min. The supernatant was collected and substrate (#45-000-947, GE Healthcare Amersham)
stored at -20 °C. For cytoplasmic and nuclear extracts, was used to image blots using the Typhoon Scanner
cells were washed with and collected in 1 mL ice-cold 9410. Image J software (National Institutes of Health)
PBS. Cells were centrifuged at 129 × g (4 °C) for 5 min. was used to obtain the mean grey intensity for the
The supernatant was aspirated and combined with bands of interest.
400 μL Lysis buffer 1 [10 mmol/L (4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid) (HEPES) (pH 7.9), 10 MTT assay
mmol/L KCl, 0.10 mmol/L ethylenediaminetetraacetic The MT T [3 - (4,5 - dimethylthiazol-2-yl) -2,5 -
acid (EDTA), 0.10 mmol/L ethylene glycol-bis diphenyltetrazolium bromide] assay was performed
(β-aminoethyl ether)-tetraacetic acid (EGTA), 1 mmol/L to determine cell viability after treatment. Fresh SFM
dithiothreitol (DTT), 0.5 mmol/L phenylmethylsulfonyl (1 mL) was added to each well followed by addition of
fluoride (PMSF), 10 μg/mL leupeptin, and 10 μg/mL 111 μL MTT. Cultures were then incubated at 37 °C
aprotinin] and vortexed vigorously for 10 s, followed for 45 min. Media was aspirated from each well and 1.5 mL
by incubation on ice for 15 min. Next, 100 μL 5.4% dimethyl sulfoxide added followed by rocking at 25 °C
igepal was added, samples vortexed for 10 s, and for 30 min. Absorbance was read at 492 nm with the
then centrifuged at 14,000 × g (4 °C) for 5 min. The Synergy 2 plate reader (Biotek Instruments).
supernatant was collected and stored at -20 °C; then
100 μL Lysis buffer 2 (20 mmol/L HEPES, 400 mmol/L NF-κB p65 binding assay
NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, Nuclear factor-kappa B (NF-κB) activation was
1 mmol/L PMSF, 1 mmol/L leupeptin, 10 μg/mL aprotinin) measured using the NF-κB p65 transcription factor
was added to the remaining pellet. The samples were kit (#89859, Thermo Scientific) per manufacturer’s
then vortexed for 15 min at 4 °C and then centrifuged instructions. Briefly, binding buffer (50 μL) was added
at 14,000 × g (4 °C) for 22 min. The supernatant was to each well, which was pre-coated with the NF-κB
collected and stored at -20 °C. binding consensus sequence. Then, 10 μL of nuclear
extract was added to each well, in duplicate. Following
Immunoblot analysis incubation for 1 h at 25 °C with mild agitation, wells
Protein extracts were subjected to 10% sodium were washed 3 times with 200 μL of wash buffer,
dodecyl sulfate (SDS) - polyacr ylamide gel then 100 μL of primary antibody (1:1,000) was added
electrophoresis (PAGE) and immunoblot analysis. to each well, followed by a 1-h incubation at 25 °C,
Briefly, protein (50 μg) in loading dye (0.25 mmol/L without agitation. Wells were washed as described
Tris, pH 6.8, 10 mmol/L DTT, 30% glycerol, 10% above and 100 μL of secondary antibody (1:10,000)
SDS, 0.05% bromophenol blue, and 50 μL/mL was added to each well, followed by 1-h incubation at
β-mercaptoethanol) was boiled for 10 min and then 25 °C, without agitation. Finally, wells were washed
loaded into gels. Electrophoresis was performed in 4 times with 200 μL of wash buffer, and 100 μL of
running buffer (250 mmol/L glycine, 25 mmol/L Tris, chemiluminescent substrate was added to each well.
0.1% SDS) at 100 V for 15 min, and then at 125 V for Chemiluminescence was measured immediately with
145 min for an overall running time of 2 h. Proteins a Nikon plate reader.
were transferred onto polyvinylidene fluoride (PVDF)
membranes in transfer buffer (195 mmol/L glycine, RNA extraction
25 mmol/L Tris, pH 8.0, 20% methanol) at 100 V for Following cell stimulation, cells were washed 3 times
90 min. After transfer, PVDF membranes were rinsed with ice-cold PBS, incubated in Trizol (1 mL/100 mm
and blocked in bovine serum albumin (BSA) in 1× dish) at 25 °C for 5 min, and then lysates were
Tris-buffered saline-Tween (TBST) (150 mmol/L NaCl, collected in nuclease-free tubes. Chloroform (0.2 mL)
25 mmol/L Tris, pH 8.0, 2 mmol/L KCl, 0.1% Tween- was added to lysates followed by manual shaking of
20-TBST) for 2 h at 25 °C with rocking. For cluster tubes for 15 s. Lysates were then incubated at 25 °C
of differentiation 68 (CD68), 2% BSA was used and for 3 min, centrifuged at 12,000 × g for 15 min (4 °C)
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ October 19, 2017 221