Hello All!
I am trying to generate HCT 116 stable cell line. I studied many protocols and proceeded as follow:
1) I cultured cells in 6 wells plates at density of 5 x 10^5.
2) At 70% confluency, I transfected the cells with the PRPF plasmid using Xfect transfection reagent by Clontech.
3) After 4-6 hours, I changed the media and kept the cells for next 24-48 hours in the incubator.
4) Afterwards, I started incubating the cells with 1mg/ml hygromycin. I changed media containing 1mg/ml hygromycin every 24 h, and continued 7-10 days. Cell density was continuously decreasing and I was expecting to obtain a colony of transfected cells, but I failed. In the last, all cells died and there was no formation of clone.
5) One time I changed the protocol and transfected the cells for three consecutive days (changing media after 6 and 24 h intervals), and then incubated the cells with 1mg/ml hygromycin. I continued for 10 days, but this also didn't work.
Please give me some useful suggestions. Thanks
Hi!
Maybe try to not change the medium every day and also (more important) decrease the conc of hygromycin which could be too toxic for HCT116 cells. In particular, after transfection, split cells in different wells of 6-well plate and add different conc of hygromycin. Then use the cells which survive to the highest conc of hygromycin.
Another thing...do you have a control plate? for example, a plate with untransfected cells which are treated with the same conc of hygromycin. Maybe your transfection did not work and that's why you could not get resistant cells. In this case try another transfection reagent like Fugene 6 which works fine with HCT116 cells
You could also try retrovirus or lentiviral transduction. These are much more efficient than stable transfection.
Take note, too, if your cell density is too high. If cells that are dying are coming off of the surface of the dish as "sheets" of cells, dying cells may be pulling transfected cells along with them. So to go with Andrea's suggested above, if you do re-plate the cells, you may wish to plate them at a lower density.
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