I need to tite a retroviral producing cell clone. I don`t how long I should incubate the cell with diluted retrovial supernant and polybrene, and if I should passage the cell before G418 selection, and if I should, how can I calculate the titer?I had done the experiment by the following procedure but the cell all died after 7day`s G418(300ug/ml) selection: 1. passage NIH3T3;2. 24h later, discard the medium, add 1ml diluted retrovirus and polybrene(to 8ug/ml);3. 24h later, add DMEM-10(G418 400ug/ml) 3ml;4. 3days later, changing the medium to fresh DMEM-10(G418 300ug/ml) and repeated this step every 3days together 3 times.The cell survived about thousand clones after 5day`s selection but all died after 7 day`s selection. Who can tell me why and how should I do?
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how to tite the retrovirus
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