I have been doing stable cell lines with knockout genes using CRISPR/Cas9 system for months. I am using ready-for-use plasmids from Santa Cruz. The first step I do co-transfection in target cell line and followed by puro selection. Then, I split cells to do single cell colony isolation to ensure bioallelic knockout. But I usually have issues at this stage no matter what kind of cells or plasmids I have used. There are something appeared in plates. They keep increasing. I could identify what they are. I have ruled out the possibility of serum precipitation or cell secretion. I tried the several rounds and changed the medium, serum, transfection reagent, selection drug etc, but couldn't solve the issue. Other cells cultured at same time never have such problems. I attached the pictures. Hope someone can help me out.
Doesn't appear like contamination. Looks like dead cells. Observing bunches of cells (clones) dying during selection is common during stable cell line selection. What is happening if you continue the selection?
it is not contamination..when cells undergo apoptosis they give rise to such structures. such structures are also visible whn u have embryoid body formation. does the knock out effect stem cell like properties to ur cells?
Thank you for your answers! I wish they would not be a contamination. I didn't keep the cultures since this kind of structure appeared more than the clones. I tried knockout several genes in different cell lines. I don't know whether the knockout effects are connected with stem cell properties. Anyway, I may start it over and continue the selection, pick the clone, expand it. Let's see what will happen.
Dear Jun! I did have that several times with my breast cancer cells MCF7 and some MDA cell lines. I did purchase a cloning rings from Sigma and cleaned the petri dish tilting it at 45ª angle with saline for several times. When done, added 10 ml of saline to check if the dead cells were gone and then proceed with the cloning. I took me a bit of time to get decent amount of cells but those that survived give me good answers. I hope that you could achieve make good stable cell lines.
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