Encyclopedia of Experiments: Immunology
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Transcript
Grow the pET-28a(+) recombinant antibodies aminopeptidase in BL21-transformed bacteria in the presence of 0.4 millimolar beta-D-1-thiogalactopyranoside in an orbital shaker at 37 degrees Celsius for 10 hours. Seed 100 microliters 0.5 x 105 Chinese hamster ovary cells per well into a 96-well plate for incubation at 37 degrees Celsius in a 6% carbon dioxide atmosphere for 18 to 24 hours.
When the cells reach 80% to 90% confluence, dilute the pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with Opti-MEM to a final concentration of 0.1 micrograms per microliter. After five minutes, mix the 50 microliters of diluted pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with one microliter of Lipofectamine 2000 and 49 microliters of Opti-MEM.
After 20 minutes of incubation, add 100 microliters of the mixture to each well of a 96-well plate containing CHO cells. At four to six hours post-transfection, replace the medium with DMEM/F12 supplemented with 10% FBS.
After 48 hours of incubation, add 400 micrograms per microliter G418 to each well to select the stably transfected cells. After 10 days of selection using DMEM/F12 medium supplemented with 10% FBS and 400 micrograms per microliter G418, sort the cells with fluorescence-activated cell sorting. Serially dilute the harvested positive cells. Seed them at an average of 0.5 to two cells per well in a 96-well plate, and culture in 37 degrees Celsius 6% carbon dioxide incubator.
