A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells
Transcription
Seed 15,000 human embryonic kidney cells in 200 microliters of Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum in each well of a flat-bottom 96-well plate, a day before the transfection. Then, incubate the plate at 37 degrees Celsius overnight at 5% carbon dioxide. Cotransfect the cells using a linear polyethyleneimine derivative transfection reagent. Dilute 0.5 microliters of DNA transfection reagent with 10 microliters of 150 millimolar of sodium chloride.
Next, dilute 0.125 micrograms of each variable heavy and light chain-expressing vectors in 10 microliters of 150 millimolar sodium chloride. Vortex all the dilutions for 10 seconds each. Then, mix the previously diluted DNA transfection reagent with 10 microliters of DNA solution. Quickly vortex the DNA transfection mixture for 15 seconds, and incubate for 15 minutes at room temperature.
Add the DNA transfection mixture dropwise on the cells, and then, gently swirl the plate. Replace the medium 16 hours after transfection, and culture the cells for five days in serum-free medium at 37 degrees Celsius.
