thawing effect or passage number? Colony formation varies..
Posted 22 November 2013 - 11:41 AM
Posted 24 November 2013 - 12:01 AM
Passaging should be done when cells are about 70-80% confluent - that's when the split ratios become less subjective. The time/cell number thing is very difficult as it would have to be done for each different amount of cells for each time, so won't find that sort of information readily.
You could be selecting for fast growing cells (or cells that detach easily) or any number of different things, but 2 passages is pretty short to be seeing this sort of change.
However, it is entirely possible that there is some sort of switch in growth type that is the result of the cells becoming confluent.
Posted 25 November 2013 - 03:21 AM
Anyway, I have the feeling I rather tend to split them at a lower density rather than higher because I want to avoid them becoming too confluent. Do you know whether splitting them eg at 60% could lead to selection of fast growing cells? Thanks a lot!
Posted 25 November 2013 - 03:28 AM
Posted 25 November 2013 - 12:13 PM
I know what you mean about the "islands" - I have a couple of lines that grow like that too. If you want really hard lines for this, try neuronal derived lines which have extremely long processes and stop growing when the processes touch another cell, so it looks like the cells are about 50% but in reality are fully confluent. The way around this is to seed at a higher density (maybe 1:4 or 1:5 splits) than you might otherwise (means you will need to split more frequently), and try to ensure that you have a largely single cell suspension when seeding. Seeding at a lower density means that you are more likely to get the islands forming, and makes it difficult again.
Selection for fast growing cells would be done by passaging very frequently and seeding at low density. I don't think you will be doing too much selection so long as they don't seem to be growing faster than they used to.
A growth curve for any cell population should be more or less the same - some sort of logarithmic curve with a lag phase to start with and a plateau at the end.
I doubt the effect will be reversible, as most of these sorts of things tend not to be, but it may be, and is quite likely to go away if you freeze the cells down.