In the past we grew cells on uncoated coverslips to monitor NF-kB activation via immunofluorescence. Recently we started to use this method again, but switched to poly-D-lysine coated coverslips to improve cell attachment. Now many fewer TNF-alpha treated cells show bright, distinct nuclear staining. The staining looks more diffuse in both the nucleus and cytoplasm. In the past 100% of cells would have bright, distinct nuclear staining. Also, infection with a virus that used to activate NF-kB in almost every cell now activates NF-kB in only a small percentage of cells and again the staining is mostly diffuse. We will try uncoated coverslips again (cells don't adhere well), but I was wondering if anyone has had a similar issue.
Thanks!
Tight attachment of cultured cells and cell lines on a glass cover slip alters signaling from the cell surface receptors, thus altering the localization and activity of a number of transcription factors, including the NF-kB. Therefore, a better way to attach cells would be to coat/immerse glass cover slips with serum and dry them before the use. This treatment may not work with all cell types. Therefore, some optimization may be needed. Alternatively, reduce the amount of poly-D-lysin for coating glass cover slips.
- Badges
- Science topic
- Similar topics
- Developmental Psychology
- Attachment