Hi A. Richard

The images are not quite clear. I suppose the problem may be due to using a higher percentage of trypsin. You should try using 0.05% of Trypsin for 2-3 minutes and neutralize it with double the amount of complete medium. Further before plating mix the cells properly so as to avoid it forming clumps.

Hope it works.

Thanks

Hi Richard, I agree with Samir about Trypsin; too long incubation of trypsin on the cells leads to morphological changes in the cells or even kills them. However, there are other reasons as well, such as Fungi /Bacterial contaminations or inappropriate handling (e.g. too much pipetting up and down the cells leads to clumping cells). By the way, make sure that 96 well plate is the most suitable plate size for your purpose! If your experimental settings allow you, use a plate with a larger surface area and make sure you are seeding a sufficient (also not too much) number of cells on it and shake them in a way that cells spread equally everywhere.

Hope next time you get nicer-looking cells!

Thank you so much for your contributions. I will adopt these modifications.

Hello Richard,

does this behavior show up in other tests as well, or only in 96 well plates? To me it looks like a reaction to a toxic environment. This could indeed be, for example, too long incubation with trypsin.

Were the plates coated beforehand? Was the same medium used? Did you test other culture dishes for comparison?

Here you can find a good manual for the standard cultivation of SH-SY5Y:

Feles, S.; Overath, C.; Reichardt, S.; Diegeler, S.; Schmitz, C.; Kronenberg, J.; Baumstark-Khan, C.; Hemmersbach, R.; Hellweg, C.E.; Liemersdorf, C. Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies. Methods Protoc. 2022, 5, 58. https://doi.org/10.3390/mps5040058

Best regards,

Sebastian Feles