Stable cell line, polyclonal and monoclonal. - Which is better? (Jun/07/2007 )
Hi,
I've finally got my stable cell lines, after a month of experimenting with the procedures. It wasn't as easy as what is written in protocols, as most parts were left out (assuming that most people would know how to go about it). E.g., 24 hrs post-transfection, the cells would normally be quite confluent, but there's no mention of what to do with this (except a few protocols did - which I'm quite thankful). So, we're left with two decisions, just add selective medium (containing G418 or geneticin) and assume that the next day, the cells will not die because of overcrowding... Or alternatively, split the cells and resuspend in selective medium.
The latter caused quite a number of cell death, presumably by the weakened membrane integrity (caused by trypsin treatment) and thus the higher susceptibility to G418 (or geneticin)? Anyway, left that for a couple of days and did not change the selective medium as frequent as recommended (Protocol recommended: change fresh medium everyday). After cells had "stabilized", only then did I commence with the G418 selection by changing fresh selective medium everyday. Somehow, a few cells got differentiated.
The former one, it's a bit alien to me. When I see the confluency of the cell I'm handling, I have the unexplained urge to want to split it. Anyway, what I had finally decided do then was follow the protocol laid out by the author (just to experiment). And the next day, after selection with G418, cells were dying (I'm not sure because of G418 or overcrowding). I ignored it and followed strictly with the protocol by adding fresh selective medium everyday. After a week, cells were still dying (I had negative control - cells susceptible to G418). Only after the second week did I see the result of selection - Transfectants were left with noticeable quantity (still proliferating) whereas negative control had the population almost completely annihilated. Great!
Now comes the next part, some suggested generating monoclonal transfectants while others did not mention such thing. I hope to get you guys to help me out as I'm quite new to this. Can I opt for "polyclonal" or is there inherent benefit in selecting "monoclonal" transfectant? To acquire monoclonal transfectant isn't easy as I'd need to "pipette" a single mildly trypsinized "colony" under microscope. Microscope is out the laminar flow and possibility of contamination is quite high in my lab if I work out the laminar flow.
Appreciate your help
I've finally got my stable cell lines, after a month of experimenting with the procedures. It wasn't as easy as what is written in protocols, as most parts were left out (assuming that most people would know how to go about it). E.g., 24 hrs post-transfection, the cells would normally be quite confluent, but there's no mention of what to do with this (except a few protocols did - which I'm quite thankful). So, we're left with two decisions, just add selective medium (containing G418 or geneticin) and assume that the next day, the cells will not die because of overcrowding... Or alternatively, split the cells and resuspend in selective medium.
The latter caused quite a number of cell death, presumably by the weakened membrane integrity (caused by trypsin treatment) and thus the higher susceptibility to G418 (or geneticin)? Anyway, left that for a couple of days and did not change the selective medium as frequent as recommended (Protocol recommended: change fresh medium everyday). After cells had "stabilized", only then did I commence with the G418 selection by changing fresh selective medium everyday. Somehow, a few cells got differentiated.
The former one, it's a bit alien to me. When I see the confluency of the cell I'm handling, I have the unexplained urge to want to split it. Anyway, what I had finally decided do then was follow the protocol laid out by the author (just to experiment). And the next day, after selection with G418, cells were dying (I'm not sure because of G418 or overcrowding). I ignored it and followed strictly with the protocol by adding fresh selective medium everyday. After a week, cells were still dying (I had negative control - cells susceptible to G418). Only after the second week did I see the result of selection - Transfectants were left with noticeable quantity (still proliferating) whereas negative control had the population almost completely annihilated. Great!
Now comes the next part, some suggested generating monoclonal transfectants while others did not mention such thing. I hope to get you guys to help me out as I'm quite new to this. Can I opt for "polyclonal" or is there inherent benefit in selecting "monoclonal" transfectant? To acquire monoclonal transfectant isn't easy as I'd need to "pipette" a single mildly trypsinized "colony" under microscope. Microscope is out the laminar flow and possibility of contamination is quite high in my lab if I work out the laminar flow.
Appreciate your help
When I am picking colonies, I locate the lolony under the microscope, circle it with a permanent pen, go back to the laminar flow and pick the colony there. Hope this helps, auldmok
Thanks auldmok for the suggestion. The cells are too small to see and it's just too difficult to circle the bottom of the plate and at the same time looking at the microscope to ascertain the spot. Hah! I'm about to work on it right now and there're yet two decisions to make. Either proliferating the cell as polyclonal or toil with picking a few colonies and assume it's monoclonal.
Would appreciate more suggestion though...
Cheers
when we select stable transfectants, we analyze both polyclonals and about a dozen monoclonals; as there are some differences between the individual monoclonals (morphology, proliferation, apoptosis-resistance etc), I think it is worthwhile to select one or two monoclonals which meet your interest, and which are used for subsequent routine analysis
Thanks The Bearer. It's much clearer to me now.