1.Determine the best concentration of antibiotics.

Different cell lines have different sensitivity to various antibiotics, so it is necessary to do pre-test before screening to determine the minimum concentration of antibiotics on the selected cells.

(1)8 cells were inoculated in 96 well plate or 24 well plate 24 hours in advance, and the Inoculated Amount grew up in the next day 25% single layer is suitable,incubating in CO₂ incubator at 37 ℃ overnight.

(2)Change the culture medium to the medium containing antibiotics, and the concentration of antibiotics increases in gradient（0, 50, 100, 200, 400, 600, 800 and 1000μg/ml）.

(3)After 10-14 days of culture, most of the cells were killed, generally 400-800 μ g / ml.The concentration can be increased by one level when cloning, and half of the screening concentration is used when maintaining.

2.The transfection is carried out according to the previous steps.

3.After 72 hours of transfection, the transfected cells were subcultured in a 6-well plate at a ratio of 1:10 and replaced with a selective medium containing the antibiotic concentration determined in the pre-test. A single cell can be seen in the 6-well plate, and a single resistant colony can be formed by single cell division and propagation in continuous culture. At this time, two methods can be used to select monoclonal.

(1)Filter paper method:After the pancreatin was immersed in the sterilized 5x5mm filter paper, the filter paper was pasted on the single cell colony for 10-15 seconds, and then the filter paper with cell adhesion was taken out and placed in the 24 hole plate for further pressurized culture.
When the cells are full in a 24 well plate, they are transferred to a 25 cm 2 culture flask, and when they are full, they are transferred to a 75 cm² culture flask.

(2)Limited dilution method:The cells were digested and diluted 10 times continuously(10^-2-10^-10)，the cells of each dilution were added to 96 well plate for culture. After 7-10 days, the single clone growing holes were selected for cloning again.

4.The expression of foreign protein in monoclonal cells was detected by ELISA or Western blot. Because of the difference of expression level of different clones, multiple clones could be selected at the same time to select the clone with the highest expression and to preserve the species.