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  • br Colony formation assay br Underlying clonogenic assay

    2020-08-12


    2.3. Colony formation assay
    Underlying clonogenic assay was performed to study about the ef-fect of fucoidan in HepG2 cancer cell growth by following the method of Buranrat et al., [25]. The precise concentration of fucoidan (0–200 μg/ml) was added into 24 h grown HepG2 cancer cells in 6-well plates (500 cells/well). Fucoidan constituted cells were washed with PBS in terms of reassigned into fresh medium for impetus of cell growth another 24 h. Subsequently, the cells were washed thrice with PBS by disposal of old medium and fixed in 100% methanol that was stained with 0.05% crystal violet for 1 h at room temperature. After washing with tap water, the colonies were counted and images regarded by in-verted microscope linked up with camera. The counted colony values were profoundly expressed in percentile.
    2.4. Wound healing assay
    This experiment was executed to study about the cell migration by the method of Buranrat et al., [25]. The HepG2 cells were allowed to grow in 6-well plates to form a cell monolayer. Subsequently, scratches were made in the monolayer cells through a sterile 0.2 ml pipette tip that was constituted with different concentration of fucoidan (0–200 μg/ml). After 48 h, the healed wound images were regarded by using the inverted microscope linked up with camera. Wound distance was calculated by comparing with untreated control cells and values were expressed in percentile.
    2.5. Flowcytometric analysis of SRS16-86 and apoptosis
    According to the manufacture’s protocol, fucoidan induced apop-tosis in HepG2 cells were scored in the array of cells staining with annexin-V FITC through the flowcytometer. Similarly, the treatment and cells preparation were performed as like as cell cycle analysis. After washed cells twice with PBS there were added 500 μl of binding buffer (5 μl of annexin-V FITC & 5 μl of PI) and kept in the dark for 15 min at ambient temperature [26]. Subsequently, cells were analyzed by flow-cytometer and expressed as a percentage of total apoptotic cells cal-culated by flowjo software.
    2.6. Detection of nuclear condensation and mitochondrial membrane potential
    Fucoidan altered nuclear condensation in HepG2 cells were per-formed by using Hoechst staining assay. The assay was conducted with maximum concentration of fucoidan (200 μg/ml) and kept incubation for 48 h. Then the cells were harvested and thoroughly washed with
    PBS and firmly fixed in 3:1 ratio of methanol and acetic acid (3:1) for 15 min at room temperature. Before the staining, fixatives were washed away carefully with PBS and stained in 5 μg/ml of Hoechst 33,342 stain for 10 min. By the condensed nuclear morphology of HepG2 cells as a result of fucoidan treatment was regarded and simultaneously SRS16-86 imaged using the fluorescence microscope [27]. Similarly, mitochondrial membrane potential (ΔΨm) of HepG2 cells consituted with maximum concentration of fucoidan (200 μg/ml) was also studied by using JC-1 staining assay. The fucoidan (200 μg/ml) constituted HepG2 cells were added with 5 μl of the JC-1 staining so-lution and incubated in 5% CO2 incubator at 37 °C for 20 min. The stained cells washed twice with PBS solution were regarded and si-multaneously imaged using the fluorescence microscope [27].
    Single cell gel electrophoresis (SCGE) was carried out by Trevigen’s comet assay kit with slight modification. Briefly, HepG2 cell suspension (1 × 105/ml) and molten LM agarose (at 37°C) were prepared in 1:10 (v/v) ratio. From there, instantly 50 μl was poured onto the comet slide pre-coated with 1% normal melting point agarose. Then, the slides were kept at 4°C in the dark for 10 min under low humidity and dust-free environment for their better agarose adhesion on the slides. The slides were placed in an ice-cold lysing solution for 60 min at 4°C. Furthermore, the excess buffer of slides was drained and deeply im-mersed in freshly prepared alkaline unwinding solution (pH > 13: 200 mM NaOH, 1 mM EDTA in 50 ml of dH2O) for 1 h at 4°C in the dark. Subsequently, the slides were placed in an electrophoresis chamber filled with alkaline buffer (200 mM NaOH, 1 mM EDTA in 1 l of dH2O) and it runs with 21 V for 30 min., Excess electrophoresis solution from the slides was drained gently and then a couple time immersed in dH2O and 70% ethanol for each 5 min. Eventually, slides were dried for 10–15 minutes at room temperature and stored with desiccant prior to scoring slides. Silver staining was performed to visualize the comets through transmission light microscope. Randomly, 50 comets per slide were visualized and analyzed by scoring of various parameters such as head, tail and tail moment under light microscope with comet assay software.