Binzhou Medical University
Question
Asked 25th Mar, 2015
Deleted profile
Why are my cells dying?
I've been having this problem for the past week now and I don't know what to do anymore. Following siRNA transfection a week ago, all cells I plated on coverslips died. I felt it was because I used a high volume of transfection reagent (But that has never been the case). So, I decided to seed a new batch onto coverslips and the same problem occurred after 24 hours. Thus, I decided to recover a new vial of low passage cells from liquid nitrogen. All cells behaved well and I went ahead to seed them onto coverslips once again. However, following siRNA transfection 24 hours ago, the cells underwent massive death once again. I'm totally confused as I've never encountered this before. What could I have done wrong.
Most recent answer
Have you considered that maybe your cell line is so sensitive to this siRNA that knocking down this gene will make cells dead? Why not lower the dose of siRNA seeded on your plate, and then see what happen.
All Answers (8)
Stockholm University
Are you knocking down the same gene as before? I have seen my cells dying when trying to knockdown genes that are important (for nutrient uptake). Also some transfection reagents are a little toxic.
National Research Council Canada, Montreal, Canada
Have you plated these cells on cover slip before? Cover slip and cell culture plate are not equivalent (these are specially coated for cell culture). Some cell lines don't attach/survive on cover slip.
University of Florida
Please provide more information on cell lines you are using. Are you seeing any contamination? As Richard mentioned, they might not like cover slips. I had issues growing some macrophage cell lines on cover slips. Do some controls and let us know how it looks. Compare them on a plate and have both lipofectamine treated and untreated cells. Also if possible compare them with scrambled or control siRNA wells. Good luck!
Thank you, Piyush. I've checked the cells (MCF7s) for possible contamination and they came out clean.
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