2.1Chemicals
DMEM, FBS, antibiotics, and all other tissue culture reagents were obtained from
GIBCO. Glutaraldehyde and osmium tetroxide were purchased from Electron Microscopy
Sciences (USA). Anti-vinculin mouse antibody was purchased from Abcam (USA). Alexa
Fluor 594 phalloidin, Alexa Fluor 488 goat anti-mouse IgG, were purchased from Invitrogen
(USA). Trypsin was purchased from Sigma (USA). CuSO4, KBr, thiobarbituric acid,
trichloroacetic acid, and other commonly used chemicals were purchased from Sigma or
Merck
2.2 Isolation of LDL
LDL was isolated from human plasma in the density range 1.019-1.063 by
ultracentrifugation as described. Human plasma was isolated from volunteer blood by centrifugation 15 minutes at 3000 rpm, 4 “C in the presence of 0.3mM EDTA followed by
ultracentrifugation at 40,000 rpm for 20 hours at 10 “C. VLDL was removed as the top layer.
Potassium bromide was added to a final density of 1.063 followed by ultracentrifugation at
40,000 rpm 10 C for 20 hours. Typically, 4 ml LDL was derived as the top layer from each
preparation of 50 ml human serum. The purity of LDL was justified by HPLC procedure
using Superdex (Waters) gel filtration column. The isolated LDL was dialyzed against PBS
to remove KBr. Protein concentration was measured by optical reading at 280nm. Typical
protein concentration was about 15 mg/ml using this procedure.
2.3Copper oxidation of LDL
Copper oxidation was performed as described in Mao et al(37). LDL and incubated at 37
“C for 4 hr. The reaction was terminated by the addition of excess EDTA followed by dialysis
in excess of PBS. TBAR assay was performed to monitor oxidation.
2.4Detection of TBARS(TBA assay)
The colormetric thiobarbituric acid (TBA) assay with minor modification was utilized to
justify lipid peroxidation. Fifty μl of sample was added to 300 μl of 20% trichloroacetic acid
(TCA), followed by the addition of 300 μl of 0.67% TBA in 0.05N NaOH and mixed
vigorously. The mixture was incubated at 80 - 90° C for 30 minutes to develop the
MDA-TBA adducts followed by a centrifugation at 3000 rpm for 5 minutes. Subsequently,
300 μ1 supernatant were transfered in duplicates onto a 96-well microtiter plate and optical
density was measured at 540nm using an ELISA plate reader
2.5 Isolation of mouse peritoneal macrophages
Resident peritoneal macrophages were isolated and cultured from 5 mice (-20g each) and
were washed lx with Dulbecco’s modified Eagle medium (DMEM) and lx with DMEM
containing 10% fetal bovine serum. The cells were added to different sizes nanodot in DMEM
containing 10% fetal bovine serum and 100 pg/ml penicillin and culture for 48 hrs at 37°C in
an incubator containing 5% CO2 with 90% humidity. The non-adherent cells were removed and the monolayers were then placed in DMEM containing 10% fetal bovine serum
supplemented with 100 μg/ml oxidized LDL or acetyl LDL and plates were further incubated
for additional 24 to 48 hrs..
2.6 Oil red 0 staining
Monolayers of macrophages prepared on nanodot surface were fixed with 10%
formaldehyde in PBS (pH 7.4) for 10 minute at room temperature, and then stained with Oil
Red 0 and counterstained with hematoxylin for 10 minutes(38).
2.7 Fabrication of nanodot arrays
Nanodot arrays were fabricated as described previously (36, 39). A TaN thin film of 150
nm in thickness was sputtered onto a 6-inch silicon wafer followed by deposition of 3
µm-thick aluminum onto the top of a TaN layer. Anodization was carried out in 1.8 M sulfuric
acid at 5 Volts for the 10-nm nanodot array, or in 0.3 M oxalic acid at 25 Volts, 60 Volts, and
100 Volts for 50-nm, 100-nm, and 200-nm nanodot arrays, respectively. Porous anodic
alumina was formed during the anodic oxidation. The underlying TaN layer was oxidized into
tantalum oxide nanodots using the alumina nanopores as template. The porous alumina was
removed by immersing in 5 % (w/v) H3PO4 overnight. The dimension and homogeneity of
nanodot arrays were measured and calculated from images taken by JEOL JSM-6500
TFE-SEM.
2.8 The cells viability assay.
Cells were harvested and fixed with 4% formaldehyde in PBS for 30 min followed by
PBS wash for three times. And membrane was permeated by incubating in 0.1 % Triton
X-100 for 10 min, followed by PBS wash for three times. The sample was incubated with
4',6-diamidino-2-phenylindole (DAPI) and phalloidin for 15 min at room temperature.
2.9 Scanning electron microscopy (SEM)
The harvested cells were fixed with 1% glutaraldehyde in PBS at 4 ºC for 20 minutes,
followed by post-fixation in 1% osmium tetraoxide for 30 min. Dehydration was performed
through a series of ethanol concentrations (10-min incubation each in 50%, 60%, 70%, 80%,
90%, 95%, and 100% ethanol) and air dried. The specimen was sputter-coated with platinum
and examined by JEOL JSM-6500 TFE-SEM at an accelerating voltage of 5 keV.
2.10 Immunostaining
Cells were harvested and fixed with 4% paraformaldehyde in PBS for 15 min followed
by PBS wash for three times. Membrane was permeated by incubating in 0.1 % Triton X-100
for 10 min, followed by PBS wash for three times, blocked by 1 % BSA in PBS for 1 hr, and
PBS wash for three times. The sample was incubated with anti-vinculin antibody (properly
diluted in 0.5 % BSA) and phalloidin for 1 hr, followed by incubating with Alexa Fluor 488
goat anti-mouse antibody for 1 hr followed by PBS wash for three times.
2.11 Quantitative real-time RT-PCR
Total RNA was extracted from macrophage and foam cell using TRI-reagent (Talron
Biotech) according to the manufacturer's specifications. The RNA was isolated using
chloroform extraction and isopropanol precipitation. The crude RNA extract was immediately
purified with an RNeasy Mini Kit (Qiagen) to remove impurities and unwanted organics.
Purified RNA was resuspended in DEPC water and quantified by OD260. The OD260-to-OD280
ratio usually exceeded 2.0 at this stage. For cDNA synthesis, 1 µg total RNA was annealed
with 1 µg oligo-dT, followed by reverse transcription using SuperScript® III Reverse
Transcriptase (Invitrogen) in a total volume of 50 µl. Between 0.2 and 0.5 μl of the reverse
transcription reactions were used for quantitative real-time PCR using SYBR Green I on an
iCycler iQ5 (Bio-Rad Laboratories). Cycling conditions were as follows: 1× [5 min at 95°C]
and 50× [20 s at 95°C, 20 s at 55°C, and 40 s at 72°C]; fluorescence was measured after each
72°C step. Expression levels were obtained as threshold cycles (Ct), which were determined
by the iCycler iQ Detection System software. Relative transcript quantities were calculated
using the ΔΔCt method. The GAPDH, were used as reference genes and were amplified from
the same cDNA samples. The difference in threshold cycles of the sample mRNA relative to
the GAPDH, mRNA was defined as ΔCt. The difference between the ΔCt of the untreated control and the ΔCt of the SMF-treated sample was defined as ΔΔCt. The fold change in
mRNA expression was expressed as 2ΔΔCt. The results were expressed as the mean ± SD of six
experiments.