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Transient Transfection Protocol


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Transient Transfection Protocol

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

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:

  1. Preheat the water bath cauldron to 37-40°C, and add 10 mL culture medium to the centrifuge tube.
  2. Take out the cell from the liquid nitrogen or refrigerator, place it quickly in the preheated water bath, and shake frozen pipes to have it uniformly heated.
  3. When liquid is completely melted in frozen pipes, pour it into the centrifuge tube with culture medium.
  4. Centrifuge 5 min and discard the supernatant.
  5. Suspend precipitate with culture medium. Cells are replated in a culturing bottle and cultivated by the conventional method.

Transfection

  1. The cultured cells are inoculated into 6-wells plates. Add 2-4 mL complete medium, mix and place in CO2 incubator, 37°C for the night.
  2. The following solutions are prepared under sterile conditions:
    • A: 2 μg plasmid is diluted with 100 μL serum-free medium.
    • B: 25 μL Lipofectamine transfection reagent is diluted with 100 μL serum-free medium.
  3. Mix solution A and solution B, let rest about 30 min at room temperature.
  4. When around 80% unilaminar cells formed at the bottom of culture flask, wash cells two times with serum-free medium and add 1 mL serum-free medium each well . Then add the solution from step 3 by drop to each well, mix gently place in CO2 incubator, 37°C for 24 hours.
  5. Pour out the transfection solution, add complete culture medium and continue culturing. 3-4 days later, the expression levels of protein are determined.

Expression detection

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.

Cell count

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:

  1. Wash and dry the cover slip of blood count, digest cells and prepare the unicell suspension.
  2. Dilute cell suspension at certain multiples.
  3. Cover the blood count with cover slip, slowly inject 10 μL cell suspension onto the blood count with transfer liquid gun.
  4. Observed cell under a microscope, count cell numbers, as shown in Figure 1.

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