Cell line and reagents
The mouse connective tissue-derived fibroblast cell line L-929 was obtained from Shanghai Fu Xiang Biotechnology Co., Ltd. (China). HyClone fetal bovine serum (FBS) was purchased from Shanghai Yu Bo Biological Technology Co., Ltd. (China). LPS (derived from E. coli O111:B4), CCK-8 (cell counting kit-8), 4 % paraformaldehyde, sealing medium and trypsin were purchased from Beyotime Institute of Biotechnology Co., Ltd. (China); MIF was obtained from R & D systems (USA); rabbit anti-mouse TLR4 antibody, FITC-conjugated donkey anti-rabbit IgG secondary antibody and donkey serum were purchased from Santa Cruz Biotechnology, Inc. (USA). Quantitative PCR kit was purchased from Wuhan Google Biotechnology Co,. Ltd. (China). PE-conjugated anti-mouse TLR4/MD2 antibody (Miltenyi Biotec, Inc.) was purchased from Shanghai Univ-bio Co., Ltd. (China).
CCK-8 assay for fibroblast proliferation assessment
L-929 cells were cultured with DMEM containing 10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5 % CO2 (Forma Series II CO2 incubator, Thermo Electron Co.). Mouse fibroblast suspension was adjusted to 5 × 104/ml, and 200 μl were seeded per well in 96-well plates. After 1 h incubation (to allow cells to adhere to plates), cells were treated with LPS at various concentrations for 48 h. Then, cell proliferation was assesses by adding 20 μl CCK-8 reagent in each well, followed by incubation at 37 °C for 2 h. Signals were spectrophotometrically measured at 450 nm on a full wavelength microplate analyzer (Molecular Device); data were recorded as optical density (OD). The procedure described above was also used to evaluate the effects of various concentrations of MIF in the presence or absence of 25 μg LPS.
Immunofluorescence for TLR4 detection
A total of 1 × 106 L-929 cells were seeded on sterile coverslips (0.13 mm thickness) in each 6-well plates, in 1.5 ml of medium. 40 h later, coverslips were gently washed 3 times with PBS and fixed with 4 % paraformaldehyde at room temperature for 20 min. After PBS wash, blocking was carried out with donkey serum (1:10) for 2 h at 37 °C; then, the coverslips were sequentially incubated with rabbit anti-mouse TLR4 antibody (1:50 dilution; 4 °C overnight) and FITC-conjugated donkey anti-rabbit IgG secondary antibody (1:100; 37 °C 1 h). The coverslips were mounted with sealing medium, followed by analysis on a laser confocal scanning microscope (Zeiss LSM-710).
Real time PCR for TLR4 mRNA expression quantitation
L-929 cells were seeded 5 × 105 per well in 6-well plate treated with MIF at three concentrations, and two replicates per concentration were applied. 48 h later, the medium in each well was aspirated, the attached cells were washed with PBS; after addition of 1 ml Trizopure, total RNA was extracted. RNA purity and concentration were assessed by OD measurement at 260/280 nm on spectrophotometer (Thermo Nanodrop 2000). cDNA was obtained with reverse transcription kit (ReverTra Ace-alpha), and real time PCR was carried out with Toyobo thunderbird SYBR qPCR Mix (quantitative PCR amplification kit). Specific primers were designed and synthesized by Invitrogen Biotechnology (China): TLR4 [NCBI(GenBank):NM_021297], forward, 5'- AGTTTAGAGAATCTCTGGTGGCTGTG-3', and reverse, 5'- TTCCCTGAAAGGCTTGGTCT-3'); β-actin [NCBI(GenBank):NM_007393.3], forward, 5'- CTGAGAGGGAAATCGTGCGT-3', and reverse, 5'-CCACAGGATTCCATACCCAAGA-3'. Thermal cycling conditions included: initial denaturing step at 95 °C for 1 min; 40 cycles of 95 °C for 15 s, 58 °C for 20 s and 72 °C for 20 s; final extension at 72 °C for 5 min. All samples were tested in triplicate on 96-well PCR plate. Relative expression of the TLR4 gene was calculated using the △△Ct method.
Flow cytometry for detection TLR4 expression on cell membrane
L-929 cells were seeded at a density of 5 × 105 per well in 6-well plate, with MIF added at 375 ng/ml for 48 h. Then, cells were pelleted centrifugation at 300× g for 10 min, and resuspended in 100 μl buffer solution. Afterwards, 10 μl PE-conjugated TLR4-MD2 (CD284) antibody was added for 10 min at 4 °C, and cells were analyzed on flow cytometer (BD Accuri C6).
Statistical analyses were performed with SPSS software (SPSS 19, Inc, Chicago, IL, USA). Data are mean ± SEM, and one-way ANOVA with dunnett's test was used for group comparisons. *P < 0.05 was considered statistically significant.