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

and then the upper aqueous phase was collected in 60.3 °C; reverse: 5’-GTT GGT TGT CTT TGA GAT
fresh microcentrifuge tubes. Isopropanol (0.5 mL) was CCA TGC CAT TG’-3’, 60.1 °C; and β-actin-forward:
added to the aqueous phase, followed by incubation 5’-GAA GGA TTC CTA TGT GGG CGA CGA-3’, 60.5 °C;
at 25 °C for 10 min, and then centrifuged at 12,000 × g reverse: 5’-GAG CCA CAC GCA GCT CAT TGT AG-
for 10 min (4 °C). Next, the supernatant was removed 3’, 60.3 °C.
and RNA pellets washed with 70% ethanol, and
centrifuged at 7,500 × g for 2 min (4 °C). This ethanol Immunocytochemistry
wash step was repeated twice. RNA pellets were Cells were exposed to SFM with or without LPS (1 μg/mL),
dried at 25 °C for 15 min, followed by addition of 35 for 10 min at 37 °C. After stimulation, cells were gently
μL nuclease-free water. RNA samples were incubated washed 3 times with ice-cold PBS and fixed with 4%
in a 65 °C water bath for 10 min and then stored paraformaldehyde (PFA) for 10 min at 25 °C. Cells were
at -80 °C. To obtain RNA concentrations, samples washed 3 times with ice-cold PBS and permeabilized
were thawed on ice and ng/mL was determined with 0.01% Triton-X in PBS for 10 min at 25 °C.
with a Nanodrop Spectrophotometer 1000 (Thermo Next, cells were washed as described above and
Scientific), and validated with the 260/280 ratios primary antibodies mouse-TLR4 (1:1,000) or rabbit-
between 1.8 and 2.0.
CD68 (1:1,000) in PBS added to respective wells
and incubated overnight at 4 °C. The next day, cells
RNA integrity were washed 3 times with TBST and incubated with
RNA gels were prepared with NorthernMax-Gly fluorescently labeled secondary antibodies, anti-rabbit-
Reagents (Ambion) to determine RNA integrity. A AlexaFluor-647 (1:1,000) or anti-mouse-AlexaFluor-555
1% agarose gel was prepared with NorthernMax- (1:1,000) while rocking for 2 h at 25 °C. Negative control
Gly buffer. To prepare samples, 3 μL RNA, 3 μL cells were unstimulated cells only incubated with
nuclease-free water and 6 μL glyoxal sample loading secondary antibodies. Cells were washed 3 times with
dye were incubated at 50 °C for 30 min. Samples TBST and labeled with [4’-6-diamidino-2-phenylindole
were loaded and electrophoresed at 100 V for 45 (DAPI)-PBS)] for 10 min, rocking at 25 °C. Finally, cells
min. RNA gels were stained with SYBR Safe (3 μL/50 were washed 3 times with TBST, cover slips removed
mL in NorthernMax-Gly buffer) and rocked on a from wells, and mounted on slides using anti-fade
Multimixer (Lab-Line Instruments, LLC-Melrose Park, gold mounting media (Invitrogen). Slides were dried
IL) for 30 min. To visualize the 28S and 18S bands, overnight and then imaged.
RNA gels were imaged on Typhoon Scanner 9410 (GE
Healthcare Life Sciences) at 450 V. Epifluorescence microscopy

Real-time-polymerase chain reaction TLR4 and CD68 immunofluorescence was imaged
RNA was reverse transcribed using the SuperScript with an epifluorescence microscope (Olympus BX51)
using Spot 5.1 software. Images were captured with
IV VILO Master Mix (Invitrogen, #11766050) and the 20× objective using fluorescent channels for DAPI
cDNA (100 ng) was used to perform real-time (350 nm), TLR4 (TRITC-555 nm), and CD68 (Cy5-
polymerase chain reaction (RT-PCR) for genes of 647 nm). Images were processed with CellProfiler
interests. The primer for CD68 was designed using cell image analysis software [20] , and quantified in
rat CD68 messenger RNA (mRNA) from the NIH GraphPad Prism 7.0.
National Center for Biotechnology Information website
and obtained from IDT Technologies. The rat primers
for TLR4, TNFα, and β-actin were obtained from IDT CellProfiler
Technologies. RT-PCR mix included 2× PowerUp All images of nuclei were processed in Image J,
SybGreen, 0.5 μmol/L forward primer, 0.5 μmol/L with global contrast settings to clean-up and define
reverse primer, and nuclease-free water up to 15 μL. nuclear boundaries for CellProfiler’s automatic
Thermocycler settings were as follows: 50 °C-2 min, nuclear recognition algorithm. A CellProfiler pipeline
95 °C-2 min, (95 °C-15 s, 60 °C-1 min for 40 cycles), was created for each fluorescent channel image
95 °C-15 s, 60 °C-1 min, 95 °C-15 s. Primer sequences set to define global pixel intensity thresholds. All
used were as follows: TLR4-forward: 5’-GAA GCT ATA image sets were then processed for high-throughput
GCT TCA CCA ATT TCT CAC AA-3’, 60.2 °C; reverse: quantification in CellProfiler to measure area and
5’-GAT AGG GTT TCC TGT CAG TAC CAA GGT TG- mean grey intensity. High-throughput quantification
3’, 60.1 °C; CD68-forward: 5’-CTC AGC AGC TCT ACC can process hundreds of images while standardizing
ATG AGG TTC-3’, 59 °C; reverse: 5’-CTT CCG GTG the recognition and measurements of each cell in
GTT GTA GGT GTC TC-3’, 59.2 °C; TNFα-forward: 5’- every image. This process increases sample size
CAG ATC ATC TTC TCA AAA CTC GAG TGA CA-3’, while removing inherent subjectivity of hand tracing.
222 Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ October 19, 2017