Transfection-Tissue culture

  • Thread starter Goodie
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  • #1
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Should I always split my cells at their mid log phase? Or does it only matter at transfection? How do I know that my cells are in the mid log phase? How do i know that my cells are 80-100% confluence? Is this 80-100% a rough estimation or accurately done?

Thanks.
 

Answers and Replies

  • #2
Sho'Nuff
Its all rough estimates, 80-100% means usually a full dish of cells, depending on the transfection methods and the cell lines used the general idea is that cells need to be around 50% confluent and preferably not in colonies for optimal transfection. I usually split my cells 24 hours before transfection.
 
  • #3
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So when my flask is confluent then my cells are at the mid log phase? The split ratio is usually 1/2 to 1/20. What decides which factor we should use? :uhh:
 
  • #4
Sho'Nuff
nope, the general idea about splitting at mid log phase is that the cells keep dividing while if you thake em at end log phase (confluent dish) the cells have stopped because they ran out of space and have to re-enter the cell cycle which presumably takes a long time (depending on cell type). So its faster if you need many cells. Personally I think this is bullocks because treating the cells with trypsin will also stop them. This then only holds true for cells in suspension and there they dont run out of space but rather out of nutrients.

If you want to know the factor I need to know the cell type..... a full dish of primary human fibroblasts will stop cycling if you split them more than 1/4 while transformen human embryonic kindey cells can go 1/15 without any problem.
 
  • #5
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Sho'Nuff said:
nope, the general idea about splitting at mid log phase is that the cells keep dividing while if you thake em at end log phase (confluent dish) the cells have stopped because they ran out of space and have to re-enter the cell cycle which presumably takes a long time (depending on cell type). So its faster if you need many cells. Personally I think this is bullocks because treating the cells with trypsin will also stop them.
So does it mean that a 80-90% confluence is most probably at mid log phase and 100% confluent is at end log phase?

Sho'Nuff said:
This then only holds true for cells in suspension and there they dont run out of space but rather out of nutrients.
What do you mean?

Sho'Nuff said:
If you want to know the factor I need to know the cell type..... a full dish of primary human fibroblasts will stop cycling if you split them more than 1/4 while transformen human embryonic kindey cells can go 1/15 without any problem.
For example why one splits a flask into two new flasks (split ratio 1/2) instead of 4 new flasks (1/4) ? Is that the amount of cells one needs that also decide it? :uhh:

Thanks.
 
  • #6
Sho'Nuff
Goodie said:
So does it mean that a 80-90% confluence is most probably at mid log phase and 100% confluent is at end log phase?
no at 80-90% most of the cells have reached confluency and some are still dividing so if the whole dish is viewed this would be near end log phase. at 100% all the cells have stopped

at 50% confluency most of the cells will still be growing so since most of the cells are now in log phase the plate can be regarded as being in log phase. mid log phase is when most cells on the plate are dividing (so at 50% or so) but note that the individual cells do not have to grow faster. Your looking at growth of the population as a whole, not the single cells

Goodie said:
What do you mean?
Basically that for cells in suspension it does matter if you split them at mid log because they will keep growing but for cells that grow attached to the plate it doesnt matter at which stage you split them because they will arrest anyway due to the trypsin treatment. (your original question was: Should I always split my cells at their mid log phase?)

Goodie said:
For example why one splits a flask into two new flasks (split ratio 1/2) instead of 4 new flasks (1/4) ? Is that the amount of cells one needs that also decide it? :uhh:
Thanks.
Not just that, some cells need to be in close contact with their neighbours to be able to grow, they depend on growthfactors of surrounding cells that are released in the medium. If you dilute them too much they will just stop.
 

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