Hey guys, I always have problems with the attachment of HEK293t cells to glass coverslips while doing IFA. They are so easy to detach from the coverslips while washing. The coverslips are already pretreated with polylysine, but it seems polylysine does not help. I used 4% paraformaldehyde to fix the cells for 20 mins at 37℃, has anyone tried a different fixation buffer that works better? I believe a lot of people might have the same problem when dealing with this type of cells, could anyone give me some suggestions? Thanks a lot!
If you coat the glass with poly-L- lysine, the cells don't attach specifically and also don't spread well in my experience. I don't know what you want to observe, but I would leave out the coating step. When you let the cells attach in medium with serum, they will spread on the glass as there are also extracellular matrix components in the serum. In defined media, I would coat the glass slides with an extracellular matrix protein. Timing is also important, you should let them adhere for at least 30 minutes but probably longer. The fixation you use is too long in my opinion, I fix the cells maximum 10 minutes at room temperature. Furthermore, the washing step should be performed carefully to not rupture the cells (I use a small syringe attached to a pump and only put it in the some corner of the well).
Dear Bing!
Please You also look at this answer:
https://www.researchgate.net/post/Would_293T_cells_adhere_to_glass_bottom_well_plates
293T cells adhere to both plastic and standard microscopic glass slide without Polylysine. However, you need to be careful at every time you wash your cells because they are not super-adherent.
If you are going to do confocal microscpy, I would recommend to seed your cells on a glass coverslide, you will get a far better resolution. I usually conduct my initial test using non-coated coverglass (to optimize antibodies, etc.) and do my ''publication ready'' image acquisition using polylysine slide (cell adherence is much better and you get a bigger cytoplasm with 293T).
Beware that 293T cells are a bit flat once they are fixed and mounted. So you won't get that many slice out of them.
If you have the money, Eppendorf and other companies have cell imaging slide with 1, 2, 4 and 8 separation that are a blast to work with. They are expensive, but well worth the money.
As an example, the 8 chamber gives you a space comparable to a 48 well plate and makes it easy to do all your staining using a minimal amount of antibodies (plus you can buy them with polylysine coating) and have most of you condition right on one slide optimized for imaging.
and article:
Article Optimized Transfection Strategy for Expression and Electroph...
Hi Bing,
HEK293T cells are very weakly non-adherent. I use retinoblastom cells which are cultured in suspension and having similar problems, although 0.1 ug/ml poly-D-lysine seems to solve attachment to glass coverslips in my case.
Have you tried precoating your coverslips using gelatin? This is a protocol I have just begun to use and I have found it works very well:
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-non-adherent-cells
This way, you can culture your HEK293T cells without worrying about attachment so much, perform your treatment and then resuspend the cells to attach to the coverslip. Below is the protocol for preparing the glass coverslips:
https://www.rndsystems.com/resources/protocols/protocol-preparation-gelatin-coated-slides-histological-tissue-sections
Let us know how you get on.
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