I am developing stably transduced HEK293T cells (lentivirus) with constitutively active RhoA gtpase. I have no experience in Microscopy and I want to characterize these cells visually and expand them prior to western analysis. I see this round morphology, but i am unsure if this is a bacterial/viral contamination or the cells are just sad. Are these just vesicles? These cells look different than HEK293T WT.
Hi Jean. If there is bacterial contamination, it can be observed very clearly by seeing the media itself. Media will be turbid in nature. As you are establishing them using Lentivirus, the morphology change may occur. You need to grow the cells continuously using the selective drug (puromycin) for 20 days minimum. Ideally, you should have an un-infected control group which should also be treated with puromycin along with the stable transfection one. This makes the comparison easier.
Thank you for your response. The media is not turbid so likely not bacteria. The RhoA+ cells + puromycin do look different than non-transduced +/- puromycin. However, one thing that I hadn't thought of was a puromycin kill curve. Perhaps the concentration of puro used altogether is causing the anomaly.
It's hard to tell exactly from the picture. But one explanation for the clumpy/rounded appearance is that the cells are dying under selection because the transfection didn't work. You might try trypsonizing, checking viability and replating the cells. Also, you do need to run a puro kill curve for every cell line you're working with. In my experience, 2 ug/mL puro is about right for HEK293T. But you should always check yourself.
Hi Diana, I re-plated the cells and they looked much happier. I even did a second round of puro selection, and the cells were robust. What size wells/plate do you use when you do the puro kill curve? I appreciate your assistance. Thanks.
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