Hello,

I tried to measure NFkB-activation by means of flow cytometry several years ago but I was not convinced by the results. The difference between NFkB-activation/ non activated cells was quite small. The method was published probably in JBC around the year 2000.

The idea is based on the fact that NFkB translocates to the nucleus when activated. First you stimulate your cells for example with LPS, then isolate the nuclei, permeabilize them, introduce the antibody against NFkB, incubate, wash, introduce the secondary fluorescence coupled antibody, wash again and fix the cells. Additionally you should add an DNA stain to discover doubletts of nuclei.

In case of NFkB-activation you should see an increase in fluorescence (depending on the conjugation of your secondary antibody) and hence, you should be able to distinguish between non activated and activated cells (nuclei).

I don't say that this method does not work at all but you have to consider a plenty of time for establishment. At least there are a lot of Parameters to be optimized: What is the time for activation, which Stimulus, how to permeabilize the cells (we used the Fix and Perm-kit), which fluorescence gives the best Signal/noise Ratio etc.

In my opinion the detection with fluorescence microscopy might be easier (however I never tried this).

best regards

Dirk

It would probably depend on what cell type you're interested in. If you're looking at doing confocal imaging, then examining nuclear translocation (i.e. NFkappaB staining colocalising with DAPI staining) gives clear and striking results, rather than looking at phosphorylation. I found decent quantitation when using log10 dose response.

However, for flow cytometry, I found the BD Phosflow NFkappaB p65 antibodies to be very good. They have quite an apparently small dynamic range in detecting quantitative changes in phosphorylation, much as the previous commentor says. However, I was able to find extremely consistent results and statistically significant changes when using the antibodies in a flow cytometric assay examining NFkappaB activation in PBMC using a patient cohort, so I think they are a good reagent.

I have used the microscopy approach, using a p65 antibody, with a red Alexafluor labeled secondary antibody (does not fade too fast), and plain DAPI for the nuclei. you can used either conventional (widefield) fluorescence microscopy or confocal to analyse this. The confocal gives a "cleaner" separation between nuclear and cytoplasmic staining than widefield. As I analysed the images using ImageJ I preferred the confocal approach. If you are just counting positive and negative nuclei manually, widefoeld is fine. The ImageJ analysis is fairly straightforward. Identify nuclei by thresholdinging on DAPI signal , erode these nuclei by a couple of pixels to properly separate from cytoplasm. Run analyze particles to get count (total number of nuclei); Detect p65 signal, and threshold (can use automated threshold such as Otsu), then do a "AND" between p65 positive image and nuclei image (both are binaries), and get object count to get number of positive nuclei.

Hello, I measured phosphorylated-p65 by flow cytometry using an antibody from Cell Signaling. We reported all materials and the protocol on our publication (Matteucci et al. Cell Death Dis. 2010 Oct 7;1:e81.). I suggest to perform a kinetic experiment from 15’-30’ up to 48h depending on different cell lines. In our model we find an initial decrease of phospho-p65 followed by an induction.

Dr. Van't Hof -- which conjugated p65 antibody did you use and from which supplier, or did you conjugate it yourself? cheers, Rob Hagan

Hi Robert, the anti-p65 was from cell signalling technology, the best one was  their #8242. We didn't use this conjugated , but did a convential two step stain using an alexafluor-594 labeled anti-rabbit secondary antibody (life technologies A-11012 ).

hope this helps,

Rob

Dr Van't Hof, could you please tell what fixation and permabilization method you use for this staining and in what kind of buffer you stain?

Thanks in advance,

Wojciech

Dear Wojciech, we basically followed the protocol for immuno fluorescence on the Cell Signalling web page. Just search for the antibody product code and it will come up.The only change to that protocol was basically the use of freshly made paraformaldehyde (4% in PBS) in stead of formalin.

In what kind of tissue did you do the IF?

I've tried with other antibodies in fixed mouse brains and it didn't work very well.. Also I found this paper that could be interesting regarding this question: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210105/