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Cell culture
and treatment of cells

The human
prostate cancer cell lines, PC3 and LNCaP (kindly provided by Eray Metin GÜLER,
Bezmialem Foundation University), were grown in Roswell Park Memorial Institute
(RPMI) 1640 medium supplemented with streptomycin (100 ?g/mL), penicilin (100
units/mL), 2 mmol/L glutamine, and 10% fetal bovine serum (FBS) and HUVEC
(kindly provided by Gürler AKPINAR, Kocaeli University, DEKART), were grown in
Endothelial Cell Growth Medium (ECGM) supplemented with Low Serum Growth
Supplement (LSGS), streptomycin (100 ?g/mL) and penicillin (100 units/mL). Cells
were maintained in 95% air, 5% CO2 at 37°C. Nobiletin (Sigma Aldrich, USA), Nobiletin
was dissolved in dimethyl sulfoxide (DMSO) to make stock solutions of 100 mM
and equal amount of DMSO was included in controls for every experiment. Cells
were treated Nobiletin at the indicated dose (20 and 40 ?M ) and time (6 and 24
h) in complete RPMI and ECGM medium. In some experiments, cells were treated
with/without Poly I:C (5 ?M; Invivogen, USA) followed by treated with Nobiletin
and analyzed. Cells that were used as controls were incubated with cell cultre only.

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WST-1
proliferation assay

WST-1 assays
were carried out according to the manufacturer’s instructions (Roche). Cells
were seeded in 96-well, culture plates at a density of 5000 cells per well in a
final volume of 100 ml per well culture medium at 37 °C, 5% CO2.
After 24 h incubation, refreshed culter medium without phenol red and with
different concentrations (0-5-10-20-40-80-160
µM) of Nobiletin. WST-1 reagent was added at a volume of 10 ml per well
and the cells were incubated for up to 3 h. Absorbance at 450 nm was measured
using a microplate reader (Thermo Scientific Multiskan™ FC Microplate
Photometer). The experiment was repeated three times. Control wells were
containing culture medium and WST-1 reagent, but no cells were included to
determine background fluorescence.

qRT-PCR

Total RNA was
isolated from cell lines using the RNA isolation kit (Vivantis, MALAYSIA)
including DNase treatment. The RNA concentration was determined
spectrophotometrically. The ratio of absorbancesat 260 and 280 nm, measured in
a NanoDrop ND-3300 spectrophotometer (Thermo Scientific, USA). First strand
cDNA was made using the cDNA Reverse Transcription kit (Vivantis, MALAYSIA)
following the manufacturer’s instructions. Expression of TLR3 gene was
determined by quantitative reverse transcription polymerase chain reaction
(qRT-PCR) using SYBR Green PCR Master Mix (Bio-Rad, USA) using the
gene-specific primers listed below and normalized to GAPDH expression. The
primers used are, TLR3 F 5? TAG CAG TCA TCC AAC AGA ATC 3?, TLR3 R 5? AAT CTT CTG
AGT TGA TTA TGG GTA 3?, GAPDH F 5? TCG ACA GTC AGC CGC ATC TTC TTT 3?, and
GAPDH R 5? ACC AAA TCC GTT GAC TCC GAC CTT 3?. All real time PCR were performed
in triplicate. Differences in TLR3 mRNA expression level, which varied with
dose and time, were compared with the Relative Expression Software Tool (REST).

ELISA for CASP8

Cysteine-aspartic
acid protease-8 (Caspase-8, CASP8) protein
levels were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) with a Human CASP8 ELISA
kit (Elabscience, USA) targeting CASP8 in cell culture supernates. LNCaP, PC3,
and HUVEC cells were cultered 10 mm petri dishes at a density of 1×106
cell/petri cells per well. After 24 h incubation, refreshed culter medium
without phenol red and serum treated as indicated with Poly I:C (5 ?M) with or
without Nobiletin (20 or 40 ?g/mL) for 6 and 24 h. Culture supernates were collected and CASP8 levels were determined following the manufacturer’s instructions. A total of 3 independent experiments,
each in triplicates, were assayed,
and the mean VEGF protein level from each
duplicate was used for statistical analysis.

Western blotting

LNCaP, PC3, and HUVEC cells
were cultered 10 mm petri dishes at a density of 1×106 cell/petri
cells per well. After 24 h incubation, refreshed culter medium without phenol
red and serum treated as indicated with Poly I:C (5 ?M) with or without
Nobiletin (20 or 40 ?g/mL) for 6 and 24 h. Total cell lysates were prepared by
lysing and scraping the cells off the petri dishes with RIPA Lysis Buffer
(Thermo Scientific) containing 1 mg/ml leupeptin and 1 mM PMSF (Sigma-Aldrich).
Protein concentration was determined by using the Bradford Assay (Bradford, 1976). 1 ml 1X Bradford Reagent (Bio-Rad, ABD)
was added 1 µl cell lysate and 19 µl standart buffer mixture and vortex. After
5 minutes incubation, samples was measured using NanoDrop ND-3300
spectrophotometer (Thermo Scientific, USA) absorbance at 595 nm. The measured
protein concentrations of the samples were calculated by comparison with the
BSA standard curve previously prepared according to 595 nm. Measurements for
each sample were repeated three times, and the results from the average of
these replicates were accepted as concentrations of protein samples. Equal
amounts of proteins (30 mg) were
subjected to SDS-PAGE and then transferred on to nitrocellulose membrane. The
filters were saturated with 5% nonfat dry milk in TBS. Mouse monoclonal Abs against total TLR3 and TICAM-1 was from
Santa Cruz Biotechnology (Santa Cruz, CA). The secondary Ab were HRP-conjugated
goat anti-mouse (Santa Cruz, CA). After incubating with the first and secondary
Abs, the membranes were washed three times for 15 min with TBS-T containing
0.1% Tween 20. Ab detection was performed by using the chemiluminescence system
(Clarity™ ECL Western Blotting Substrate, Bio-Rad, ABD).

 

MMP-9 and MMP-2 gelatin zymography

Activity of MMP-9 and MMP-2
was determined in culture supernatants by gelatin zymography. PC3, LnCaP and
HUVEC cells were cultered 10 mm petri dishes at a density of 1×106
cell/petri cells per well and treated as indicated with Poly I:C (5 ?M) with or
without Nobiletin (20 or 40 ?g/mL) for 6 and 24 h. Culture supernatant was
electrophoresed in 8% SDS-polyacrylamide gel electrophoresis (PAGE) containing
0.1% gelatin (Sigma-Aldrich Co). Gels were soaked in 2.5% Triton-X100 for 2
hours, followed by incubation in digestion buffer (10 mM CaCl2, 50 mM Tris, pH
7.4, 1% Triton-X100) for 20 hours at 37°C. The gels were then stained with
Coomassie Brilliant Blue R-250 (0.2% in 40% methanol, 10% acetic acid) and
destained in 20% methanol, 10% acetic acid solution. Clear bands representing
MMP-9 and -2 activity were imaged. Clear bands analyzed for MMP-9 and MMP-2
activity with ImageJ software.

Statistical analysis

Statistical assessment
was carried out with the program SPSS version 22.0 for Windows (SPSS, Inc, Chicago,
Illinois, USA) ImageJ (Image Processing and Analysis in Java) and REST (2009
V2.0.13). The results were analyzed using one-way analysis of variance (ANOVA) and post hoc test (2-sided Dunnett’s t) to test both overall differences and specific differences between each treatment and control. Differences in the level of TLR3
mRNA expression varying with dose and time were compared with the REST
statistical program. With the ImageJ program, the densities of the bands
obtained as a result of gelatinase zymography were measured in 3 repetitions. Results
were expressed as mean ± standard error of mean
(SEM). p<0.05 was considered significant.

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