10 replies to this topic

[justwonder]

should i add antibiotica :

1. in medium after thawing?
2. in freezing medium?
3. in incubation of transfected cells?

or should i add antibiotica only when my mammalian cells have a vector with the appropriate selectable marker for that antibiotica?    

thanks.

Edited by bioforum, 14 March 2005 - 07:09 PM.

[ladybug]

justwonder, on Mar 14 2005, 03:42 PM, said:

should i add antibiotica :

1. in medium after thawing?
2. in freezing medium?
3. in incubation of transfected cells?

or should i add antibiotica only when my mammalian cells have a vector with the appropriate selectable marker for that antibiotica?   

thanks.

I was always taught to put the antibiotics in the freezing medium.  My basic freezing medium recipe is:
   56% basic medium, 40% FBS, 5% DMSO, 1% antibiotics.  This may change for your particular cells but it's a start.

[justwonder]

Quote

I was always taught to put the antibiotics in the freezing medium. My basic freezing medium recipe is:
56% basic medium, 40% FBS, 5% DMSO, 1% antibiotics. This may change for your particular cells but it's a start.

what if your cells have no vector with the appropriate selectable marker, would you still add antibiotica? would not the cells die of it, since it have no resistance against the antibiotica?

thanks.

[fred_33]

justwonder, on Mar 14 2005, 11:42 PM, said:

should i add antibiotica :

1. in medium after thawing?
2. in freezing medium?
3. in incubation of transfected cells?

or should i add antibiotica only when my mammalian cells have a vector with the appropriate selectable marker for that antibiotica?   

thanks.

hi

for freezing cells i use serul 10%DMSO or serum free medium +10%DMSO and it works very well. It's not necessary to add antibiotics like geneticin or puromycin.

But if you mean penicilin streptomycin by "antibiotics" you can add it in the medium after thawing. mammalian cells are normally resistant to these classical.

But NEVER add antibiotics during transfection

hope i as helpful.
Fred.

Edited by fred_33, 15 March 2005 - 01:19 AM.

[justwonder]

Quote

for freezing cells i use serul 10%DMSO or serum free medium +10%DMSO and it works very well. It's not necessary to add antibiotics like geneticin or puromycin.

in order to add geneticin or puromycin, should my cell have vector with marker which is resistant to these antibiotica? because the cells will die if it does not have resistant marker, won't it? or the cells are naturally resistant to these?

Quote

But if you mean penicilin streptomycin by "antibiotics" you can add it in the medium after thawing. mammalian cells are normally resistant to these classical.

you mean naturally resistant to and not that we have already transfected it with a resistant vector against these antibiotica?

Quote

But NEVER add antibiotics during transfection

i understood.  

[fred_33]

hi
strepto and penicilin are bacterial antibiotics and mammalian cells are normally resistant. But they are sensitive to Puromycin and neomycin(which one is also called G418 or geneticin). Resistance to this two antibiotics generally comes with the vector you used for transfection.

But for puromycin, it's quite delicate. In fact, some cell lines are immortalized after a transfection of a vector encoding an immortalizing protein (E6 or large T-SV40 antigen). And it's possible that for selection of these immortal cells, the manufacturers had used puromycin. So before using cells, you should take informations about her resistance/sensitivity regarding antibiotics you want or may use..

Fred

[justwonder]

thanks, Fred.

So in summary; add only antibiotica if the cells are naturally or artificially (vectors etc) resistant to them. Correct?

[fred_33]

hi
i'm ok with that. you're right. but if your cells are resistant to puromycin whithout adding a vector with resistance marker, that mean that all your cells are immortal and in this case you do not need to add puromycin. you will do your selection after the transfection with the selective marker present on your vector.

i wish you good experiments and succeding.
fred

Edited by fred_33, 15 March 2005 - 03:06 AM.

[justwonder]

Quote

i'm ok with that. you're right. but if your cells are resistant to puromycin whithout adding a vector with resistance marker, that mean that all your cells are immortal and in this case you do not need to add puromycin. you will do your selection after the transfection with the selective marker present on your vector.

  can you explain this? immortal cells are not resistant at the same time, are they? it would clear my mind alot. thanks.

[fred_33]

hi
i know that there are cell lines that are immortalized by expression of a particular protein . This protein expression is possible by transfection of a gene in a cell which wasn't immortal at the beginning. This transfection was realized with a vector that could contain a selective marker (ex puromycin, cause it's a quick selection) For a quick selection of transfected cells, cells that are now immortalized, the selection is made by adding an antibiotic (in our example it would be puromycin).

The company / the researcher soon get the immortalized transfected cells.

For YOUR experiment now :
the cell is resistant to peni strepto (mammalian cell)
the cell s resistant to puromycin (immortalization step)

so for your experiment, the selective marker should be other than puromycin (generallly neomycine).

i hope you may understand my poor explanations. I'm sorry to be so unclear.

[justwonder]

Thank you very much, Fred. You have helped me alot with this understanding. I could never do it myself. I truly appreciate your great help.