I am using G418 to generate a stable cell line. My cells reached 90% confluency before massive cell death occurred. I was just wondering if it would be recommended to split the cells and continue selection, or whether it would be better to leave the cells for another day and hope massive cell death kicks in soon.
The real problem is that the cells destined to die will huddle together and thus avoid dying! Also when they do die, because they die in a few large cohorts and they tend to release all sorts of nasty stuff that that affects the remaining cells, including the G418 resistant clones, so some of them die too! The best thing to do is before you do your transfection is to do a kill curve with G418 such that all the cells that are going to die will be dead before they reach confluence. Also if you can try using puromycin resistance as your selectable marker because the cells die much faster (less than a week in most cases). All this is easy to say in hind sight, and it does not help you much here and now!.
If I was going to select my clones via ring cloning, I would persevere with the cells at, or approaching confluence, but I would change the media every day, sometimes twice if I fed them when I first got in, and then saw that there were loads of floaters or rounded up refractile cells. Also, when feeding them squirt the media you are about to remove around the dish just to make sure that some cells on the verge of detatching come off as well.
If I were cloning by limited dilution I would split them first thing in the morning and re-plate (in media containing G418), and then look at them in the evening, and if it is obvious that whilst some cells have attached, but the majority are floaters, then I would re-feed. The problem with this strategy is that the colonies which have been formed by particular clones will now be dissociated, so when they are all re-plated many of the clones will become duplicated. I would then expand the culture and freeze a pool which you could then go back to, and select clones in G418 on about 10 96 well plates, plating out about 1 cell every 4 wells, and leave them for about 10 days. After that carefully look at the wells and check which wells have colonies in , and when they have grown to colonies which you can just see by eye, passage them on into a 24 well plate and expand them. However you must remember that there will always be a few clones that are duplicates, so when you screen them, pick cells with different morphologies or expression levels, so as to maximise the chance of getting as many different clones as possible. I would also keep all my clones in G418 until I had expanded and screened them, and frozen down a few aliquots. After that I would thaw them in G418 for a few days, then grow them on in G418 free media, do a few experiments for a limited number of passages and then throw them out.
good luck!
Well the massive cell death is normal: these are the cells which do not have stably incorporated the construct. Cells do not die immediately but rather after some time, and very suddently, and all at once. Remaining (few) cells are the one you selected and having stable expression of the construct. Spliting cells is therefore not recommended since it is not a problem of confluency.
Please read this for further information:
http://www.lamondlab.com/pdf/stablecells.PDF
Thanks a lot for this info. Just to confirm though, I do not have massive cell death as of yet and the cells are confluent. Would you recommend just leaving the cells even though they are confluent and just wait for massive cell death, or starting again with a lower initial plating density?
Much appreciated.
Usually the problem is not the plate density, it is the G418 concentration. Each cell type has a different response range to the antibiotic, and if they do not die, then they are much more resistant to G418 rather than just too numerous. Still it takes some time (1-2 weeks) before figuring this out.
The real problem is that the cells destined to die will huddle together and thus avoid dying! Also when they do die, because they die in a few large cohorts and they tend to release all sorts of nasty stuff that that affects the remaining cells, including the G418 resistant clones, so some of them die too! The best thing to do is before you do your transfection is to do a kill curve with G418 such that all the cells that are going to die will be dead before they reach confluence. Also if you can try using puromycin resistance as your selectable marker because the cells die much faster (less than a week in most cases). All this is easy to say in hind sight, and it does not help you much here and now!.
If I was going to select my clones via ring cloning, I would persevere with the cells at, or approaching confluence, but I would change the media every day, sometimes twice if I fed them when I first got in, and then saw that there were loads of floaters or rounded up refractile cells. Also, when feeding them squirt the media you are about to remove around the dish just to make sure that some cells on the verge of detatching come off as well.
If I were cloning by limited dilution I would split them first thing in the morning and re-plate (in media containing G418), and then look at them in the evening, and if it is obvious that whilst some cells have attached, but the majority are floaters, then I would re-feed. The problem with this strategy is that the colonies which have been formed by particular clones will now be dissociated, so when they are all re-plated many of the clones will become duplicated. I would then expand the culture and freeze a pool which you could then go back to, and select clones in G418 on about 10 96 well plates, plating out about 1 cell every 4 wells, and leave them for about 10 days. After that carefully look at the wells and check which wells have colonies in , and when they have grown to colonies which you can just see by eye, passage them on into a 24 well plate and expand them. However you must remember that there will always be a few clones that are duplicates, so when you screen them, pick cells with different morphologies or expression levels, so as to maximise the chance of getting as many different clones as possible. I would also keep all my clones in G418 until I had expanded and screened them, and frozen down a few aliquots. After that I would thaw them in G418 for a few days, then grow them on in G418 free media, do a few experiments for a limited number of passages and then throw them out.
good luck!
First of all, you have to design a growth curve of such cell line and you'll discover a lot of answers of a lot of questions that still in your mind.
Cells heve to be subconfluent to respond to G418. You have to determine the dose at low density of cells. Subculturing 24 hours after tarnsfection to dilute out the cells makes the untransfected respond to antibiotic.
"split" the cells, but realize that many of the cells are dead and dont know it, meaning that the high density of cells is keeping them alive. If you trypsinize and replate, that will likely solve your problem, because the dead ones wont sit back down....Good luck
Some cells are resistant to G418, and make sure your cell lines are suitable for this experiment. Of course, cell density is another problem. G418 is toxic for dividing cell, so you may need to dilute your cells as they have well dividing.
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