Mei et al.
FA-97 Ameliorates Colitis via Antioxidantion
colon of mice was immersed in 4% paraformaldehyde (pH Myeloperoxidase (MPO) and Inducible
7
.4) for 24 h, embedded in paraffin, cut into 4 µm sections
Nitric Oxide Synthase (iNOS) Activity
Measurement
For MPO and iNOS activity measurement, 0.5 cm colonic
using standard histological techniques to prepare paraffin
sections (for further analysis, e.g., Immunohistochemistry and
immunofluorescence assay). The prepared paraffin sections
were stained with hematoxylin and eosin (H&E), observed
and photographed with a bright-field microscope (Leica
Microsystems, Heerbrugg, Swiss).
◦
samples stored at −80 C were taken from the distal region
and cut into pieces. Colon pieces were homogenized in ice-
cold potassium phosphate buffer (100 mmol/l, pH = 7.4),
sodium orthovanadate (10 mM), PMSF (100 mM) and protease
inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) with a
TM
FastPrep 24 homogenizer, and centrifuged at 12,000 rpm/min
◦
Single-Cell Preparation and FACS Analysis
The distal colon tissues (0.5 cm) were taken, cut into 5 mm
pieces, transferred into 50 ml conical tubes and incubated
in 20 ml in Hanks’ balanced salt solution (10% FBS, 100
U/ml penicillin, 100 µg/ml streptomycin and 2 mM EDTA)
for 30 min at 4 C. The supernatant was taken to assess the MPO
and the iNOS activities according to manufacturer’s instructions.
O-dianisidine method was used in the MPO activity assessment
to quantify the neutrophil infiltration into inflamed colonic
mucosa. The MPO activity assay kit and the Nitric Oxide
Synthase Assay Kit were purchased from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China). Protein concentration
of each sample was determined by bicinchoninic acid (BCA)
protein assay kit (Thermo, MA, USA). MPO and iNOS activities
were measured by a standard curve of samples in units of
MPO/mg or iNOS/mg of protein.
◦
at 37 C, 250 rpm/min for 1 h. Cell suspensions were passed
through a 70 µm filter and the remaining colon tissues
were washed with PBS, cut into 1 mm pieces and incubated
with Hanks’ balanced salt solution (5% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin and 1 mg/ml type VIII
collagenase) at 37 C, 250 rpm/min for 30 min, and vortexed
for 20 s at the start, middle and end of incubation. Cell
◦
suspensions were passed through a 70 µm filter, pelleted by
◦
centrifugation at 1,500 rpm/min for 10 min at 4 C, washed Immunohistochemistry
with PBS, and then diluted down to 5–10 × 106 cells/ml.
For FACS analysis, single-cell suspensions were washed in
FACS buffer (PBS, 0.1% BSA) and stained by the following
panel of monoclonal antibodies to the cell surface molecules:
allophycocyanin (APC)-conjugated anti-CD11b (clone M1/70,
catalog # ab25482, Abcam) purchased from Abcam, Inc.
Distal colonic segments (0.5 cm) from mice in each
group were taken and the colonic paraffin sections were
prepared as described above (Histological analysis). The
immunohistochemistry assay used for testing the expression
of IL-1β, IL-6, TNF-α, p65, c-Jun, HO-1, and Nrf2 of the
colonic tissues was performed according to the manufacture
instruction in the Immunohistochemistry Application Solutions
Kit (#13079, Cell Signaling Technology, Danvers, MA, USA).
Briefly, colonic paraffin sections were deparaffinized by being
incubated in sequence in three washes of xylene for 5 min each,
two washes of 100% ethanol for 10 min each, two washes of
95% ethanol for 10 min each, and then washed twice in dH2O
for 5 min each. Bring slides to a boil in 10 mM sodium citrate
buffer (pH = 6.0) and maintain at a sub-boiling temperature for
10 min. Cool slides on bench top for 30 min and wash three times
for 5 min each. After being incubated in 3% hydrogen peroxide
for 10 min, sections were blocked with 100 µl blocking solution
for 1 h at room temperature and stained with the following
primary antibodies: rabbit monoclonal anti-IL-1β and anti-p65
antibodies (1:100, Cell Signaling Technology, Danvers, MA,
USA); rabbit monoclonal anti-IL-6, anti-TNF-α, anti-c-Jun,
anti-HO-1, and anti-Nrf2 antibodies (1:200, Abcam, Cambridge,
United Kingdom). After incubation in a humidified chamber
(Cambridge, UK), fluorescein isothiocyanate (FITC)-conjugated
anti-F4/80 (clone BM8, eBioscience) and phycoerythrin (PE)-
conjugated anti-Gr-1 (clone RB6-8C5, eBioscience) obtained
from eBioscience (San Diego, CA, USA). Cells were washed
in FACS buffer and detected using an LSRII flow cytometer
(Becton Dickinson, Franklin Lakes, NJ). Data were analyzed
using flowjo7 software (Tree Star, Ashland, OR). CD11b F4/80
+
+
+
cells appeared to be monocytes/macrophages, and CD11b Gr-
1+
cells appeared to be monocytes/neutrophils, which accords
with the previous study (23).
Immunofluorescence (IF) of Colon Tissues
Immunofluorescence analysis was performed to test the CD11b
positive inflammatory cell infiltration in colon tissues. Briefly,
paraffin-embedded colon tissue sections were prepared as
described above and followed by being deparaffinized, rehydrated
and washed in PBS. After being treated with 3% hydrogen
peroxide and blocked with 5% BSA, the colon tissue sections
were incubated at room temperature with FITC-conjugated anti-
CD11b (1:100, clone M1/70, catalog # ab8878, Abcam) for 1 h.
Then the slides were then counter-stained with DAPI (1:1000). at
room temperature in dark for 30 min. The reaction was stopped
by washing in water gently for 3 min. The confocal laser-scanning
microscope (Olympus, Tokyo, Japan) was used to acquire images.
Settings for image acquisition were identical for control and
experimental tissues.
◦
overnight at 4 C, the tissue sections were washed with PBS
three times, covered with 2 drops SignalStainꢀ Boost Detection
R
Reagent for 30 min at room temperature, and washed three
times with PBS for 5 min each. Apply 200 µl SignalStain
ꢀ
R
DAB to each section for 8 min, immerse slides in dH2O and
washed twice, and then dehydrate sections by incubating in
95% ethanol for 10 s twice, repeating in 100% ethanol for
10 s twice and in xylene for 10 s twice. Colon sections were
observed and photographed with a bright-field microscope
(Leica Microsystems, Heerbrugg, Swiss).
4
Bai, Xue
