Hello everyone,
I am doing the immunofluorescence assay. And I want to see the NF-KB p65 translocate from cytoplasma to nuclear. But in my MOCK treated group, p65 still in the nuclei and very obvious. So in my INFECTION treated group, the change seemed not obvious. I tried several times to improve the protocol, by shorten the Triton-treated time, or blocking with 10%BSA et al. Still the same results and problem, The antibody is Santa Cruz and is suitable for IF, which I checked in.
Now I do not know how to do with this assay, and do anyone give me any advice is much helpful!!
NF-kB activity is very sensitive to the presence of growth factor in the media and/or stress. It would be helpful to know if you are starving your cells by reducing serum concentration prior immunostaining.
I assume you are using SC-372 from Santa Cruz. This is an excellent antibody for doing p65 IF in many cell types, but I have found that the performance depends very much on your IF processing protocol. I have not had luck with formaldehyde fixation and this antibody at all, nor Tween-20 or Triton X-100 permeabilization as the initial perm. I have, however, found a protocol that works every single time to give me good, clear results:
This protocol will work. I have tried it in two separate institutions with 4 different cell lines and have had students under my mentorship do it independently without issue. Plus, the entire protocol can be done in less than 3 hours because of the 37C incubations, meaning you don't have to worry about overnight steps.
Some other points of concern:
- Do you have positive controls? Good examples are 1 hour incubation with 1 µg/mL E. coli LPS or with 1-10 ng/mL TNFa.
- Are you imaging by confocal or widefield? Occasionally you might some low level signal in the nucleus because of out-of-focus light from other imaging planes.
- How many or what percentage of cells do you see translocated in the controls?
Edit: Because of the methanol fixation, you will not be able to do anything with the cytoskeleton on these cells. So, no phalloidin or anti-tubulin staining. Most other things should be fine.
we have used the NFkb repoter cell line from imgenex,and followed their protocol for NFkb actvity assay
I think what you see in your control condition is the basal activity of NFkB. p65 shuttles back and forth from cytoplasm to nucleus like an oscillatory pattern, influenced by activating & inhibitory signals.
To get a good answer to your biological question, you will need to synchronise the NFkB signaling in your cells and subsequently analyze their pattern.
colleagues from my lab have used TNFa in HepG2 to get synchronization (see link) but there are other ligands/stimulants as well. A pilot experiment will show you what works best.
http://onlinelibrary.wiley.com/doi/10.1002/hep.24314/full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531459/
Try using j774 cell line or mouse peritoneal cells to see true effect I think raw cells are not true picture of normal macrophages I did same work using j774 cells and had no issues in my results
As Samreen said, J774 are a much more functional macrophage cell line and in my opinion preferred over RAW, but both cell lines exhibit similar NFkB nuclear translocation dynamics. I have done this experiment with both cell lines numerous times. I would not necessarily go out of your way to try a new cell line for the purpose of seeing NFkB translocation.
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