Method for Constructing Stable Cell Lines

1. Antibiotic susceptibility testing

      1.1 Take out the cells from the carbon dioxide incubator, observe the cell growth status under the microscope, count after trypsin digestion, 2*10^5/well, spread on a 24-well plate, culture at 37°C overnight, concentration The default settings are: 0 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 10 μg/ml. Different cells have different sensitivity to antibiotics. If no suitable puro is tested, it can be improved. puro gradient concentration.

      1.2 According to the growth status of the cells, replace the complete medium every 2-3 days, and add the corresponding concentration of puro, continue to observe the cell status, and generally determine the effective killing of non-transfected cells within 4-6 days Minimum drug concentration for cells.

2. Viral fluid infected cells

       2.1 First transfect the target plasmid, Package the lentivirus and collect the virus fluid.

       2.2 Take out the cells from the carbon dioxide incubator, observe the cell growth status under the microscope, and count after trypsin digestion. The amount of cells is 2~4*10^5/well, and spread on a 12-well plate. Add 50ul of virus liquid and replace with fresh medium every other day and culture for 24 hours. Add the puro drug concentration determined to effectively kill non-transfected cells to screen out the transfected cells. At this time, the cells are polyclonal cells. And verify whether it is overexpressed. The verification experiments for each cell line are different, and the experiments should be carried out according to the actual situation.

3. Monoclonal screening

       3.1 Polyclonal cells will be monocloned after verification Screening can be done by limiting dilution method, dilute to 50 cells/10ml in 96-well plate, 100μL per well. After monoclonal proliferation, verification is performed, and monoclonal cells with higher expression levels are selected for expansion, culture, and cryopreservation.