I am preparing for a proliferation assay where I have infected KP230 cells with shScr, shCol18a1#35, and shCol18a1#37 viruses. These shRNA viruses are made with the pLKO.1 vector and have puromycin resistance. I am at the stage of antibiotic selection, 48 hours after infection, where I will use 0.2 ug/mL puromycin for each of my three samples. Overall, the three 6 cm plates will have 18,000 KP230 cells, 0.6 uL puromycin, and 3 mL 10% growth media. I am advised to wait another 48 hours after antibiotic selection before typsinizing and seeding for the proliferation assay (growth curve).
Why does antibiotic selection take 48 hours? My protocol applies to other cell lines too such as HT-1080, STS-109, and STS-148 cells, so is 48 hours a standard among most cell types? What about using a different antibiotic on other resistant genes? Can anyone explain biologically what happens during these 48 hours with time stamps? What happens if I only give it 24 hours? I appreciate all your responses in advance.
2 to 7 days in general, up to 14 days is also possible. An antibiotic kill curve experiment is required before start selection with any cell line and any antibiotics the first time. There is no strict rule how many days you shall select your cell. It depends on cell line and antibiotics and purpose of the selection. You could use lower concentration with longer selection or higher consentration with shorter selection to achieve similar goals, though the former is preferred with less cytotoxicity by antibiotics. The best way for your assay is to add a non transfected control as indication of efficient selection, which should show most cell death after 48hours. If not, you may need to treat it longer or increase the concentration of puromycin.
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