I'm trying to create a stable cell line of NSO cells that express my protein into the supernatant. I've transfected the cells, seeded them at various densities into 96 well plates and screened the wells that had single colonies colonies for protein expression using ELISA. After selecting the colonies with highest absorbances, i've expanded them into 24 well plates, and now I'm performing the second round of screening, also with ELISA. The problem is that after I expanded these colonies and screened them, the absorbances were much lower than what I observed from these colonies when they were screened from the 96 well plates. I let the expanded colonies become confluent before I screened them the second time, so is this normal? I find it very strange that the absorbance levels would decrease so dramatically. Any help is appreciated.
Hi Jason,
I think that grown cells at confluences might increase protein degradation. An optimal density of cultured for protein expression should be determined. Test culturing the cells at the same confluence used in 96 well plates to compare.
Did you continue adding selective antibiotic in the culture? Is possible that the expression plasmid was lost in cells confluent.
Maybe this link help you:
http://es-ar.invitrogen.com/site/mx/es/home/References/gibco-cell-culture-basics/cell-culture-protocols/maintaining-cultured-cells.html
Bye,
Gabriel
Hi Gabriel,
Thanks for the advice. I'm not sure measuring the OD would help in my case, the media has FBS present so OD would give me a measure of total protein from serum as well as expressed product. The media used during seeding into 96 well plates contained antibiotics (pen/strep) as well as selection reagent (zeocin), and the same media was used during expansion of the colonies into 24 well plates. Seems highly unlikely that the cells would lose the plasmid at this point because those that do, would be killed by the zeocin, and the expanded colonies are still growing.
J
Hi Jason,
Using transfection you transfer high copy numbers of your vector, in my experiments for example 10.000-100.000 copies per cell. This amount will degradually decrease over time during cell division. Only stable clones will keep the Transgene expressed, but quite often at lower level compared to transiently transfected cells due to lower copy numbers. For stable clones you need 4 - 6 weeks with passages for keeping the Cells proliferating.
Silencing might be an issue. Sometimes this is stronger, the next time all clones are expressing at high levels over a long time.
Bye,
Dennis
Hi Dennis,
Thank you for the insight, this makes sense. This is the first time I've attempted to make a stable, high-expressing cell line, so I wasn't sure these results were normal. After screening the clones from the 24 well plates, some clones still produced signal in the ELISA, while others produced virtually no signal. It appears that the clones still producing signal (albeit much lower now) are those that have stable integration of the expression plasmid. It's been just over 4 weeks since transfection, so I plan to continue to expand these clones into 6 well plates, and I'll assay expression again by ELISA. Hopefully, I will end up with several clones that still produce my protein at a high level.
Thanks for the help!
J
Hi Dennis,
I have another question that maybe you could help with. After running the ELISA on the expanded clones in the 24 well plates, I then further expanded the high producing wells into 6 well plates with fresh media (+Zeo). I expanded a total of 36 clones into the 6 well plates and after the weekend, only ~8 of the 36 clones appear to have living cells. It seems that massive cell death has occurred with the other 28 clones. For the clones that had dead cells, I went back to look at my 24 well plates from which these clones were expanded. All of those wells had dead cells in them too. Why would this be happening? Before expansion to the 6 well level, these clones had been subjected to selective media for over 4 weeks. Why would they all of a sudden start to die? Any ideas?
Thanks,
Jason
Dear Jason,
Is is possible to get low expression levels when expanded from 96 wells to 24 wells.
Initially you may see good expression levels, but most of the clones will not be able to retain the similar expression levels depends on the site of integration of plasmid. Most of the high yield clones (during the screening process) will not be stable.The medium expression level clones will be more stable. You can try to screen as many clones as possible which will help you to retain stable clone.
Cell death in 6 well plates - expression levels may change in this step . Cell death could be due to improper maintenance ( time of media change, pH, other supplements etc).
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