A scratch test is one of the most popular methods of classical cell migration assay in a monolayer culture. At the same time, the scratch assay has some disadvantages that can be easily corrected. Aim. Optimization the existent scratch assay on the base of detection of scratch wound surface area and the length of the field of observation which is more objective and less time consuming. Methods. Scratch assay. Results. The modification of scratch assay enables to perform measurement more accurately and rapidly. This approach is more simple and eliminates the main disadvantages of the classical method. Conclusions. The procedure of scratch wound width measurement calculated on the base of detection of cell free area and the length of the field of observation is more effective than the classical wound healing assay. It will be useful for the estimation of cell migration dynamics in monolayer culture for a wide range of live cell based experiments.
Aim.To generate the p85-S6K1 knockout MCF-7 breast cancer cell line and to evaluate the effect of p85-S6K1 on cell growth, migration and survival under stress conditions. Methods. CRISPR/Cas9 genome editing, Western blotting, immunofluorescence staining, MTT assay, in vitro scratch assay. Results. We generated two clones of the p85-S6K1 knockout MCF-7 cell line and tested their survival upon hydrogen peroxide treatment as well as the proliferation and migration rates. The generated cell clones display an impaired ability to survive under oxidative stress, exhibit inhibition of cell growth, cell motility and downregulation of rpS6 phosphorylation on Ser235/236/240/244 under cell starvation compared to the control cells. Conclusions. The p85-S6K1 isoform could be involved in modulation of cancer cell behaviour promoting cell growth, migration and survival. The obtained clones can be further used to study the participation of different S6K1 isoforms in the control of cell function. K e y w o r d s: mTOR/S6K1 signaling, CRISPR/Cas9, p85-S6K1 isoform.
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