I'm attempting to single-cell sort U2os cells into a 384-plate for imaging purposes, but my cells refuse to grow. I see no growth in any of the wells after 10+ days in +37 °C with 5% CO2. I can see a single cell has been successfully sorted into the wells, but it looks dead and there's cell debris on the bottom.
I detach the U2os cells from the bottle with PBS + 10 mM EDTA and change into FACS buffer for sorting, which is PBS + 10 mM EDTA + 10% FBS. I've pre-pipeted 100 µl of conditioned DMEM+10% FBS (1/3 ratio of used/fresh media) with Pen-Strep in the wells before sorting.
Any ideas what I could do differently? Or is there a more robust cell line that I could use, that has well defined organelles and is good for imaging.
10mM EDTA for cells for extended periods of time is quite harsh. I would suggest reducing the EDTA concentration to 1mM and wash it out and sort the cells in PBS/FBS only. Alternatively, you could use a reagent like Accutase, which is gentle and not very toxic to cells.
Some cells prefer to grow in large numbers rather than as individual cell clones. To mimic the presence of other cells, you could use an irradiated feeder cell line, or add conditioned media to your sorted plates.
Hi
things that you can try
1) not use the sorter. Limiting dillution: plate 0,25 cells per well. This should lead to clonal cell line
if you want to use the sorter
2) use 50% conditioned medium in the initial,step of single cell cloning.
3) during the sort use a larger nozzle (and low speed...). This will reduce the stress on the cells
4) include 5 and 10 cells per well. based on the number of clones that grow out you can statiscally say whether they are clonal.
When I w as s young, Hanks was used in moving cells, but not when growing them.?????
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