I am working with SHSY-5Y and using NEST-made tissue culture plates. I am looking for some companies that may support stronger adherence to these poorly adherent cell type.
Hello Sanjukta Ghosh
You may use Nunc flasks with Nunclon Delta treated surface.
Nunclon Delta is a cell culture–treated surface modification that makes the polystyrene surface of culture vessel more hydrophilic, promoting maximum adhesion for a broad range of cell types. This tissue-culture surface is offered across multiple formats for adherent cell culture applications. You may want to refer to the link provided below for more information.
https://www.thermofisher.com/in/en/home/life-science/cell-culture/cell-culture-plastics/nunc-adherent-cell-culture-surface.html#:~:text=Nunclon%20Delta%20is%20a%20cell,your%20adherent%20cell%20culture%20applications.
Alternatively, you may treat culture ware surfaces with extracellular matrix proteins such as collagen, fibronectin, laminin, or other factors to facilitate attachment. For instance you may coat the surface of the plate with collagen IV at 10ug/ml.
Best.
I remember working with this cell line and they are high maintenance diva cells if they want to be. We would grow undifferentiated cells on uncoated (TC treated) plates to 60-90% confluency then for differentiation (RA) we would put them on laminin-coated plates that we made in the lab.
To make the plates: coat TC plates with Poly-Ornithine at 10ug/ml in sterile water and leave in the hood overnight (12-24 hours) with the UV light on. Next day, wash the plates 2x with sterile water. Add laminin at 5ug/ml in DPBS and place in 37C cell incubator for at least 5 hours. The next day, wash plates 2x with DPBS. For 10cm plates add 12ml DPBS, seal with parafilm, and store in 4C. I think these would be good for 2 weeks to 1 month so we'd make a lot at once since it took 3 days.
**Don't let the plates dry out, wash and coat 3-4 at a time to prevent drying out**
When adding cells to plates: remove DPBS and add normal growth media and place at 37C to warm up. Add media very gently to not disturb coating - dropwise or on the sidewall very slowly. Once the cells are ready then add them dropwise to the plates with media. Then change to differentiation media the next morning.
I also remember you couldn't split them too sparsely, they like to communicate with each other so perhaps try seeding them heavier and expanding them more conservatively.
I know methods sections don't normally mention surface coatings but search through the literature and see if you can find some articles that do mention it or from groups your PI knows and you can ask them.
https://www.accegen.com/recent-posts/sh-sy5y-cell-line-culture-protocol-and-research-applications/
Thanks Julie Ann Dougherty . I am currently using collagen coated plates for my undifferentiated cells. Can I apply a laminin coat on these plates, do you think it is advisable?
My experiment protocol involves giving washes to the layered cells, laminin coat will improve the cell loss, I suppose?
I would think adding the laminin coating on top of the collagen would be ok, just might be expensive but if those are your normal plates and you have them to use then give it a try.
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