Antibiotic selection and screening for targeted cell clones in adherent culture

Forty-eight hours post transfection in adherent cells, selection with Hygromycin B in growth medium at 500 μg/ml (and for plasmids carrying the IgG genes also Puromycin at 1 μg/ml) was initiated. The selection medium was replaced every 2 days until colonies formed. At this stage, colonies were picked by carefully scraping patches of cells with a pipette tip and transferred to 24-well tissue culture plates for clonal expansion. The clones were sequentially expanded from 24-well to 6-well tissue culture plates. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, GmbH) as per the manufacturer’s protocol. Clones were further maintained without antibiotic selection in the culture media. Clonal cell lines were generated by serial cell dilution from the picked colonies.