Cell Culture
Macrophage cell line RAW 264.7 obtained from National Cell Bank of Iran (NCBI). Cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Life Technologies, Ghent, Belgium), 100 U/mL of penicillin and 100 µg/mL of streptomycin. The cells grown until reached 70–80% confluence in the culture flaks (Fig. 1a). The cells passaged and plated at 1:3 or 1:2 dilutions every 2–3 days using 0.25% trypsin and 1mM EDTA (Invitrogen LT, Merelbeke, Belgium). The cells frozen in 90% DMEM with 10% DMSO (Merck, Darmstadt, Germany) in liquid nitrogen.
Cell viability screening by MTT assay
RAW 264.7 macrophage cells seeded in 96-well plates (1×104 cells/well) and irradiated in the Radiotherapy Department of Imam Hospital at room temperature, using a Co-60 gamma-ray source (Source Canada, Theratron 780 E) with a dose-rate of 0.4 Gy/min, at a distance of 1 m from the source. This study aimed at investigating the effect of gamma radiation with 4, 6, 8 and 10 Gy doses. In control group, cells received no radiation.
Cell viability measured by MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide) assays, 24h after radiation, as described previously [19]. The percentage of cell viability was calculated and compared to control (100% of viability).
Apoptosis process assay by Flow cytometry
RAW 264.7 macrophage cells seeded in 6-well plates (16 ×104 cells/well) and irradiated with at least to 4, 6, 8 and 10 Gy doses. Other irradiation conditions done as previous assay. Control group, received no radiation. 24 h after treatment, cells collected in 500 µl Binding Buffer (Annexin V-FITC/PI apoptosis detection kit, Jiangsu Keygen Biotechnology). Annexin V fluorescein isothiocyanate and propidium iodide (FITC; 5 µl) added to cell suspension and incubated for 15 min at room temperature in the dark. The cells analyzed with FACSCalibur flow cytometer with the BD CellQuest™ Pro Analysis system (BD Biosciences, USA).
Cell cycle analysis by Flow cytometry
After collection the cells seeded in 6-well plates, the cell pellet washed twice with PBS buffer and fixed in a cold 70% EtOH solution at 4°C for 24 h. Fixed samples washed with PBS and stained with a 1 ml propidium iodide cocktail (50 µg/ml PI + 20 µl RNase A + 1 µl triton x-100) for 30 min at room temperature. Afterwards, the samples subjected to cell cycle description by flow cytometry (BD Biosciences, USA). Cells in G0/G1, S, and G2/M-phase determined by filtering for doublets and aggregates.
In vitro migration assay
For this assay, 4×104 cell/well seeded onto 24-well plates. After overnight incubation at 37°C and 5% CO2, cells starved for 24 h. A scratch then made on the monolayer using a sterile 200 ll-pipette tip. The monolayer was rinsed three times with and placed in DMEM with 1% FBS followed by irradiation (4, 6, 8 and 10 Gy). Reduction in the scratch areas monitored by inverse light microscope (ECLIPS 80i with WG filter, Nikon, Tokyo, Japan) equipped with digital camera. The images were taken at the interval time of 0, 24, and 48 h [20]. The percentage of migration calculated based on the reduction in scratch area measured at the specific time as described below:
= ((Scratch area at 0 h - Scratch area at specific time)/ Scratch area at 0 h)*100
Three-dimensional movement assay
Trans-well migration assay performed to measure three-dimensional movement of the cells under treatment condition. The cells (4×104 cell/well) were plated in DMEM supplemented with 1% FBS in the upper chamber of 8 µm pore, 24-well trans-wells plate (SPL life science Co., Gyeonggi-do, Republic of Korea). The cells allowed to adhere for 2 h then exposed to irradiation (4, 6, 8 and 10 Gy). After 24 h rest in the incubator, the polycarbonate membrane removed. The cells that migrated through pores to lower surface rinsed with PBS, fixed in 100% methanol and stained with ethanol-based crystal violet solution. Relative to control migrated cells shown as the means of the cell numbers in fifteen randomly selected vision.
Stimulation inflammatory pathways of Raw264.7 Cells with LPS
Lipopolysaccharide (LPS) (1 µg / ml) used to stimulate and activate inflammatory pathways in Raw264.7 Macrophage cells. The cells incubated for 18 h with LPS at 37 ° C, 5% CO2 and 95% humidity.
Cytokine releases assay by ELISA
Cytokines secreted from the cells to the media, determined by ELISA kits (BD Biosciences, San Jose, CA, USA), according to the manufacturer's instructions. Briefly, RAW264.7 macrophage cells (2×104 cells/well) cultured in a 48-well plate with DMEM containing 10% FBS, incubated at 37°C and 5% CO2 incubator. The content level of IL-1β, IL-6 and TNF- α in culture media evaluated at 450 nm in a BioTek ELx808 microplate reader (control, LPS and irradiated with 4, 6, 8 and 10 Gy groups). The absorbance values converted to concentrations of IL-1β, IL-6, and TNF-α (pg/mL) using standard curves prepared with serial dilutions of recombinant IL-1β, IL-6, and TNF-α standard protein.
NO and PGE2 production
After 24 h cell rest from the irradiation (4, 6, 8 and 10 Gy) cultured cells in the 6 well plates (16 × 104 cell/mL), the quantity of nitrite as indicator of NO release to the culture medium measured. The level of nitrite - stable metabolite of NO - measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid). Briefly, 100 µL of cell culture medium mixed with 100 µL of Griess reagent. Subsequently, the mixture incubated at room temperature for 10 min then the absorbance measured at 540 nm by microplate reader. Fresh culture medium used as a blank in every experiment. Quantity of nitrite determined using sodium nitrite standard curve. The concentration of PGE2 in cell culture media determined using a PGE2 assay ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) following the manufacturer's instructions.
Quantitative analysis of gene expression
Total RNA extracted from each one of the groups (control, LPS and irradiated with 4, 6, 8 and 10 Gy groups) using RNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s recommendations. Quantity and quality of extracted RNA determined by UV spectrophotometry (Eppendorff, Germany). cDNA synthesis performed from 500 ng DNase-treated RNA samples with a Quantitect Reverse Transcription Kit, using oligo (dT) primers. The specific primers used for PCR reactions (Table 1). q-PCR reaction performed using Master Mix and cyber green in an Applied Biosystems, StepOne™ thermal cycler (Applied Biosystems, USA). PCR program started with an initial melting cycle for 5 min at 95°C to activate the polymerase, followed by 40 cycles of melting (30 Sec at 95°C), annealing (30 Sec at 58°C) and extension (30 Sec at 72°C). The quality of the PCR reactions confirmed by melting curve analyses. Efficiency of primers determined for each gene using a standard curve (logarithmic dilution series of cDNA of each sample). The reference gene (GAPDH) and target genes amplified in the same run. The target genes normalized by reference gene (that had equal expression in all runs) and expressed relatively to calibrator.