SiHa cells cloning

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    • #11862
      jappy
      Participant

      Hello everybody.
      I have tried to clone SiHa cells from a stable transfection, to obtain cell colonies, each one from a single SiHa cell. I have used the dilution cloning method both on culture plates and 96-microplates chambers. I have even tried to enrich dilution culture with conditioned medium from pre-cultured cells, but all attempts haven’t get success.
      Conversely HeLa cells, which were transfected with the same plasmid, don’t have problem for cloning.
      Anyone can help me? Anyone know how to solve a similar problem? Thanks.

    • #93024
      biohazard
      Participant

      So, the problem is that you don’t get clones at all? Do the SiHa cells grow normally prior to cloning? Is the transfection ok, the reporter genes give you a signal and the cells divide and stay viable in culture?

      Maybe there’s something in your cloning protocol that kills the SiHa cells or drives them to apoptosis. Perhaps you should simply try to find an alternative protocol or cloning method. For example, if you have an access to a cell sorter, you could try that in cloning.

    • #93082
      jappy
      Participant

      Stably transfected SiHa cells grow "normally" at high cell densities. Problem arises when I try to clone by growing at very low cell densities.
      The transgene contains Green fluorescent protein open reading frame, which is expressed, but it isn’t toxic for SiHa cells.
      I believe cells are well transfected, since cells were selected with G418, although some cells don’t express GFP.
      I think the limiting step is growing at high dilution (low densities, equivalent to 50-100 cells per 60 mm plate).
      Is it possible to get single cells by FACS?

    • #93092
      JackBean
      Participant

      Do not the cells produce some growth factor, which is at low concentration, when diluted?

    • #93127
      jappy
      Participant
      quote JackBean:

      Do not the cells produce some growth factor, which is at low concentration, when diluted?

      Probably yes, but even when I put medium from precultured cells (same cells or another cells), cell growth doesn’t improve.

    • #93128
      biohazard
      Participant

      With some other cells I have succesfully used gamma irradiated (3000 to 6000 rad) cells to help in cloning. The dose is high enough to kill all the irradiated cells, but they stay alive for several days, which is usually enough for the cloned cell to start dividing and make their own "friends" and thus they don’t get killed because of loneliness 😉

      In case you do not have access to any proper radiation source (many medical haematology labs have one, for example), you could try some drug that works in a similar manner. Treat the "helper" cells with something that irreversibly halts the cell cycle and finally kills the cells and then collect the growing, untreated clones!

    • #93129
      biohazard
      Participant
      quote jappy:

      Is it possible to get single cells by FACS?

      If you have a proper sorter (such as BD FACSAria or Beckman Coulter’s MoFlo XDP) then, yes.

      But the problem with dilution probably remains the same. But check my previous post for a possible solution.

    • #93149
      jappy
      Participant

      OK. All tips are very interesting. It will be more comlex than I think, but it’s OK. Thanks a lot.

    • #93304
      jappy
      Participant
      quote JackBean:

      Do not the cells produce some growth factor, which is at low concentration, when diluted?

      Probably yes. Even, at high cell density these cells grow with slowly rate.

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