7 replies to this topic

[than4]

I know that I should "regularly" thaw out new cells so that my results do change due to mutations, genetic drift in the culture etc.  

However, how often is "regularly"?  And do you go by how many times you have passaged the cells or how long (in time) since they were thawed?

[goldfinger]

than4, on Mar 31 2010, 01:44 AM, said:

I know that I should "regularly" thaw out new cells so that my results do change due to mutations, genetic drift in the culture etc.  

However, how often is "regularly"?  And do you go by how many times you have passaged the cells or how long (in time) since they were thawed?

For 293 cells, I usually pass them about 30 times and them thaw a new patch.

[rkay447]

than4, on Mar 30 2010, 11:44 PM, said:

I know that I should "regularly" thaw out new cells so that my results do change due to mutations, genetic drift in the culture etc.  

However, how often is "regularly"?  And do you go by how many times you have passaged the cells or how long (in time) since they were thawed?

Each cell line is different.  For CHO and HeLa I go to about 25-30 before thawing but sometimes I'll see changes and problems earlier.  I frequently test by IF for mycoplasm and when my cell get to higher passages I'll see nuclear bridging.  What cell lines are you working with?  Perhaps then people who are familiar with that specific line can help answer your question.

[goldfinger]

rkay447, on Mar 31 2010, 12:55 PM, said:

than4, on Mar 30 2010, 11:44 PM, said:

I know that I should "regularly" thaw out new cells so that my results do change due to mutations, genetic drift in the culture etc.  

However, how often is "regularly"?  And do you go by how many times you have passaged the cells or how long (in time) since they were thawed?

Each cell line is different.  For CHO and HeLa I go to about 25-30 before thawing but sometimes I'll see changes and problems earlier.  I frequently test by IF for mycoplasm and when my cell get to higher passages I'll see nuclear bridging.  What cell lines are you working with?  Perhaps then people who are familiar with that specific line can help answer your question.

Hi, can you explain what is nuclear bridging and why this is bad?

thanks

[rkay447]

Nuclear bridging is when I'll see a small string of DNA between two cells by DAPI staining.  This means that when the cell divided the DNA was not properly divided and the cells did not undergo complete cytokinesis.  I often see this in high passage cells that are also starting to get many binucleated cells.  Overall, this means the cells are starting to undergo genetic mutations/crisis and it's time to thaw new cells.  I see the nuclear bridging more in CHO cells than HeLa or U2OS (these are the main cell lines I work with).  HeLa just tend to go binucleate and slow way down in their divisions.

[goldfinger]

rkay447, on Mar 31 2010, 01:41 PM, said:

Nuclear bridging is when I'll see a small string of DNA between two cells by DAPI staining.  This means that when the cell divided the DNA was not properly divided and the cells did not undergo complete cytokinesis.  I often see this in high passage cells that are also starting to get many binucleated cells.  Overall, this means the cells are starting to undergo genetic mutations/crisis and it's time to thaw new cells.  I see the nuclear bridging more in CHO cells than HeLa or U2OS (these are the main cell lines I work with).  HeLa just tend to go binucleate and slow way down in their divisions.

Thank you very much, that's very helpful

[than4]

thanks for all your feedback.  I thought somewhere between 20-40 passages would be most people's answers.

I am using choriocarcinoma cells, JEG-3 and BeWo as models of trophoblasts in the placenta.  

Looks like it's time to get some new cells out of the -80.

[bob1]

than4, on Mar 31 2010, 01:42 PM, said:

thanks for all your feedback.  I thought somewhere between 20-40 passages would be most people's answers.

I am using choriocarcinoma cells, JEG-3 and BeWo as models of trophoblasts in the placenta.  

Looks like it's time to get some new cells out of the -80.

You should store your cells in liquid nitrogen (vapour phase is best), at -80 they will not survive very long, a year or two at the very most and even then they will have had significant selection pressure from the storage conditions.