I am looking for a dilution protocol to make a monoclonal cell line from NIH-3T3 cells. Basically, what I would like to know is:
1.) When you have a single cell isolated what growth medium+supplements do you use?
2.) How often do you change the medium?
3.) How long do you culture the cells before you can see growth?
Thanks in advance!
One of the difficulties with clonal isolation and outgrowth is that most vessels have such a large volume that the cell cannot condition the medium itself. By reducing the volume in the growth environment, the effective concentration of secreted condition molecules is increased. I have not done 3T3 isolation for monoclonal line generation, but I have described a simple protocol for isolation of single cells in the paper below. It is very quick and the consumable (Terasaki microtest plate) is quite inexpensive. If you continue to use normal growth medium and change it every 1-2 days, to avoid evaporation. Once the cell has expanded to ~50-100 cells, I think moving to a 96 well plate would be fine, and then expand to a 48, 24, 12, 6 well plate from there, etc.
Article A convenient, optimized pipeline for isolation, fluorescence...
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