Hello,
I am currently working towards neuronal differentiation through the use of rNSCs. I keep running into the same problem, cell clumping. The controls (complete media) clump further than the experimental groups (neural differentiation media). I have read that this might be a result of cell debris or free DNA. I have tried to modify my culturing protocol; however, the same outcome is repeated.
Has anyone run into a similar problem? if so, how was it resolved?
Thank you in advance.
Have you tried a DNAase treatment? Can you post images of the cells?
I have not. I am fairly new at working with the cells, so my initial guess was that there might be something wrong with my culturing protocol. Most of what I have read talks about a DNAase treatment, but as I am new to this cell type, I would like to see if this has worked well with my cell type. I have attached two DAPI images.
It is a little difficult to tell without phase contrast or DIC images, but I think you are looking at normal cell island formation in the differentiation medium. Is your protocol like this one? https://www.cellapplications.com/sites/default/files/documents/instructions/Instructions%20RNSC.pdf
look at the datas/applicatins tab on that website, too.
Here is a DIC image, it has been difficult to obtain a clear image of all the cell components, or perhaps I am not as experienced in taking DIC images.
It is similar, I am following Thermo Fisher's protocol. Perhaps it is normal, as I said, I am new at working with this cell type.
Formation of islands of NSCs,as shown in your latest image, in differentiation medium is normal. For best DIC imaging the medium level is important (you don’t want a meniscus effect). If using multiwell plates stay away from the edges. Make sure you’re in focus and the condenser is adjusted before rotating the polarizer and analyzer filters.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running