I am establishing a monoclonal cell line transfected with my transgene. I have picked up the clones which are growing in 48-well plates. However, I see that there are some needle like structures as well in almost all the wells. Could anybody please tell me what could these be and how to get rid of them and how much could they impact the cells. The cells are Drosophila S2 cells.
Thank you
Ikram
Dear Dr Rao
Thank you very much for your reply. Here I just took a couple of photographs under the light microscope using my phone camera. I hope you can come to some conclusion.
I would agree, looks like media components precipitating/ crystallising. Possibly humidity is too low?
Dear Dr Hampshire and Dr Lilley
Thank you very much for your in-puts. Yes, I also have come to the same conclusion that these may be crystals forming due to evaporation of the media. These are Drosophila S2 cells which do not require to be grown in presence of Co2 or high humidity. However, I have started using more media and replenishing the media every 3 days.
Dear Ikram, can you give a quick description on the protocol you are using for cloning of S2 cells? I have to say that I've tried serial dilution which never works for me because S2 do not grow at low density, so eventually I've succeed with old school soft agar cloning technic and irradiated feeder cells. BTW the photos you showed indeed crystal from the medium. Best, Eugene
Dear Eugene
By stating "cloning S2 cells" do you mean transfecting S2 cells or what?
Hi, by cloning I mean making a monoclonal population from single cell (transfected or not)
Dear Eugene
Well, it took me quite a lot of time to establish monoclonal cell line. I used various strategies in parallel. Once was diluting the cells so much to have nearly 1 cell per 100ul and then pipetting 100ul in each well of a 96-well plate, you have to then look at each well under the microscope to check and mark the wells with only one cell for further process. Secondly, I diluted cells to nearly 1000 cells in 7ml media in a 10 cm petri dish, let it grow until they form colonies. In the second approach most of the cells die and if you are lucky some may survive, however, you could increase the number of cells to 1000 or 2000. Thirdly, I seeded 50,000 cells in a flask in 13ml media (the best strategy), within a couple of weeks you will get clones which are discrete, some may be very close to eachother but do not pick them. Pick the discrete clones carefully and transfer them into a 48-well plate, one clone per well with sufficient amount of media, let them grow for a week to 10 days and then start expanding them.
Hope this helps.
Thank you
Ikram
Dear Ikram, Thanks! Soft agar cloning is kind of laborious even though it worked for me. So I'll try dilution again next time. But how you picked clones from the flask? According to my experience they attached quite strong and oicking up might be a problem?
Best, Eugene
Dear Eugene
They don't attach so strong to make it difficult for picking. You just have to push the colony with the tip a little and the pipette up carefully without disturbing the surroundings. Also, could you please let me know about this soft agar process, I don't know how that works. May be in future it might come handy.
Thank you
Ikram
Dear Ikram: here the description https://books.google.de/books?id=HBBblgZvOA4C&pg=PA165&lpg=PA165&dq=s2+cells+drosophila+soft+agar+cloning&source=bl&ots=S-zEdrZQen&sig=IaU3DrqsjrhZECiT9dPCKhOA_GU&hl=en&sa=X&ved=0CCUQ6AEwAGoVChMI9sGGrvXpyAIVQskaCh1I1wnu#v=onepage&q=s2%20cells%20drosophila%20soft%20agar%20cloning&f=false
Hello, I tried to grow the S2 cell as well and got the same problem (needle like crystal).
Is there any way to make the crystal disappear? Will adding a few micro liter of ddH2O will do?
Thank you
Dear Shella
Just increase the amount of media. Also, keeping changing the media every 4-5 days. That should be enough. It actually happens due to crystallization when there is smaller amount of liquid.
- Badges
- Science method