Hi,
I have some problems with SH-SY5Y cells. I seeded 20000 cells/well in a 96-well plate and had no problem, but now I have a problem in seeding my cells in a 6-well plate. I seeded different number of cells (600000, 500000, 400000, 350000, 300000, 250000 and 200000) in 6-well plate, but I see cell overcrowded in 6-well plate and you can see SH-SY5Y are under stress their shape is completely rounded. In addition, I use DMEM/F12 1:1, FBS 10% and 10micro/500 ml Na2So3 (to increase GPX4 activity). I am working about ferroptosis and GPX4 and I once isolated RNA from 400000 cells/well and was performed q-pcr but I didn’t see any expression of GPX4!!. Also, I used protein lysate of this cells for western blot and I didn't see any specific GPX4 bound!!! what should I do?? I attached photo of SH-SY5Y cells in 6-well plate and my western blot membrane.
Thank you so much for your attention
Parisa
I would first try to seed the SH-SY5Y neuroblastoma cells at a lower density 0.1 x 106 in 6-well plate. Followed by extraction of RNA at 80-90% confluency (~ 1x106).
As for the qPCR results, what are the Ct values of your target gene GPX4 and what are the Ct values of your housekeeping gene(s)? I normally recommend using 2 housekeeping genes (ACTB, GAPDH).
As for the Western blot, was the antibody validated prior to running your experiment via not only WB, but perhaps also IP, IHC, ELISA? The expression of your target protein is negligible. Increasing the amount of total proteins up to 40-50ug would perhaps improve your results.
Hey Parisa, I had a similar problem with the seeding density in a 6 well plate especially if you culturing the cells over incubation periods, such as 24-48 hours.
600 000 cells/well is high especially if the growth is regularly fast. I seed at 1x106 for a 6 well plate, it should be a lot better and not over confluent preventing cell death.
I hope this helps.
Best of all!
Hi Sash and Selim,
Thank you so much for your answers. It was so useful for me.
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