Transwell chambers, equipped with a 6.5 mm diameter polycarbonate filter insert (pore size 8 μm) (Becton Dickinson, Labware, Oxford, UK), were pre-coated with 50 μg/insert of Matrigel. Cells were seeded at a density of 2 × 104 cells/insert. After incubation for 48 hours, the cells that invaded through the Matrigel were quantified using a crystal violet staining assay (Sigma-Aldrich).
Crystal violet staining assay
Manufactured by Merck Group
The Crystal Violet Staining Assay is a laboratory technique used to quantify the biomass or viability of adherent cells in culture. The assay involves the staining of cells with crystal violet dye, which binds to cellular DNA and proteins. The intensity of the stained cells is then measured using a spectrophotometer, providing a direct correlation to the number of cells present.
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2 protocols using crystal violet staining assay
1
Matrigel Invasion Assay
2
Growth Inhibition Assay of HCC Cells
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Crystal violet staining assay (Sigma) was performed to further assess the specific growth inhibitory effects mediated by Ad-ΔB/TRAIL and/or Ad-ΔB/ING4 on the human HCC cells (Hep3B and HuH7). In addition to that, this Crystal violet staining assay was also employed on normal human liver (WRL68) cells to predict the safety profile of each tested therapeutic strategy. During the assay, Hep3B, HuH7 and WRL68 cells were individually cultured at about 60–70% confluence and then treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 at various MOIs of each viral vector: 0, 1, 2, 5, 10, 20 MOIs for Hep3B or HuH7 cells; and 0, 5, 10, 20, 50, 100 MOIs for WRL68 cells. Seventy-two hours post-treatment, the culture media of each well was removed and replaced by adding 500 μl of crystal violet solutions (0.5% crystal violet in 50% methanol) for staining process. After 30 min, the wells were smoothly washed with distilled water, naturally dried, then imaged and documented as photographs. The experiments were repeated three times; and PBS-treated cell groups were concurrently analyzed as negative controls.
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