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Mammalian cells have the function of protein folding and post-translational modification, and their proteins have natural activity. There are two ways to produce proteins through mammalian cells: transient transfection and stable transfection. The main characteristic of transient transfection is rapid expression in the short term, and enough to satisfy Mini-preparation of protein. While, stable transfection can produce large amounts of protein in the long-term by constructing stable cell lines. Transient transfection means that the constructed plasmids are introduced into mammalian cells in some ways, but the foreign genes of the plasmid are not integrated into the genome of the mammalian cell. With the growth and division of cells, foreign genes will be gradually and totally lost. So plasmids can exist for 3-4 days in cells. Around this time, foreign genes can be transcribed and translated into proteins. Transient transfection has been widely used for rapid expression protein with high activity in the short term.
cell recovery is the process of thawing and re-culture cells which are stored at liquid nitrogen or -80°C. The key of cell recovery is fast which is to prevent the cell damages during thawing. The general steps of cell recovery are as follows:
Collect cultured cells, break up the cells through ultrasound or enzymatic hydrolysis, and supernatant is obtained by centrifugation. The detection of mRNA expression level and protein expression level are conducted. The cDNA need analyses with real time-PCR or reverse transcription PCR. And proteins are extracted for western blot analysis.
The principle of cell count is to obtain the total concentration of cells by determining the number of cells per unit volume. It is widely used in cell culture and testing cell growth curve. We can use blood count to count cell numbers. The steps are as follows: