What time point after treatment are you looking at? Generally speaking NF-kB activation peaks between 30-60 min after exposure to the stimulus. Also, are you sonicating your cells to be sure you are breaking open the nuclear envelope? The NF-kB antibodies I've used detect a phosphorylated subunit which is shuttled into the nucleus after activation. You'll get much less signal if you don't break open the nuclear membrane. If you are detecting a phosphorylated component, keeping the lysate cold and adding a phosphatase inhibitor may help. TNFa is also one of the most common activators of NF-kB, 10 ng/mL will usually produce a strong activation, if you want an additional positive control.

It is possible that Nf-kB pathway is not activated with LPS challenge. Why don't you try a compound which induces ROS? It also could be the amount of cells used, some detection kits needs the use of 6x10^6 or 10x10^6 cells. Try to increase the amount of cells used.

I've tried 1 X 10^6 and 10 X 10^6 cells. Both also not giving any results. After quantifying with Bradford assay, it shows that the concentration of cell lysate was high enough for the ELISA test. However, there is no sign of NF-kB activation after testing with the kit. I am going to try using H2O2 to activate and see if there is any activation on NF-kB or not.

I worked with the I worked with the same cells using 10-100 LPS ng /ml and saw induction of NO by Griess reagent

What time point after treatment are you looking at? Generally speaking NF-kB activation peaks between 30-60 min after exposure to the stimulus. Also, are you sonicating your cells to be sure you are breaking open the nuclear envelope? The NF-kB antibodies I've used detect a phosphorylated subunit which is shuttled into the nucleus after activation. You'll get much less signal if you don't break open the nuclear membrane. If you are detecting a phosphorylated component, keeping the lysate cold and adding a phosphatase inhibitor may help. TNFa is also one of the most common activators of NF-kB, 10 ng/mL will usually produce a strong activation, if you want an additional positive control.

Did you try other activators like TNF?

Hi,

To detect NFkB-mediated NO production, I used to treat 200,000 RAW plated in 12-weel plate and using DMEM 10% low endotoxin medium without phenol red, which makes a huge difference for these cells compared to BMDM. I used to treat with 100ng/mL for 24 hours. Here, if you try to evaluate the quantity of NFkB protein after your treatment, you might need to work in this time range. If you want to determine the activation of NFkB, as Eric said, you should consider shorter time. Indeed, the activation of NFkB is characterised by IkB phosphorylation, degradation and NFkB nuclear translocation.

Alternatively, you may consider qPCR to check NFkB RNA content to assess NFkB gene activation, NFkB western-blotting in nuclear extract or whole cell extract to investigatethe protein quantity and the translocation. Or IkBalpha phosphorylation with proteasome inhibitor (MG132).

Hope this help

Good luck

Check your LPS. I would try fresh LPS and see if you get the same results. LPS should be stored at concentrations of a minimum of 1 ug/uL. I have had trouble before with the efficacy of LPS. I now store at 2 ug/uL and it is better.

Did you check NO production in the medium? It is very easy to detect the nitrite level with Griess reagent. If NO is produced i can say that there is no problem regarding LPS. You should have a good stimulation with 1 microgram/ml concentration. I worked with LPS from different sources (E. coli, Salmonella) and experienced that stimulation varies. If LPS stimulates your cells then you have to see NFkB activation in comparison with unstimulated cells. I did not check NFkB activation with ELISA. I dont know which protocol you follow. You can check your cell extract preparation, do you use nuclear extract? How do you lyse the cells? You can try to check NFkB activation with transfection assay or western blot (for cytosolic and nuclear p50,65) if you have the agents or proper antibodies of course. Good luck:)

Hi everyone. Thank you so much for all the comments.

I've already checked for NO production before proceeding to NF-kB. Indeed, there is NO production after challenging the macrophage cells with 1 microgram/mL of LPS.

For the ELISA test for NF-kB, I'm using p65 Activ/NF-kB kit from IMGENEX. Yes, I am using the nuclear fraction for the NF-kB test and I've challenged 1 X 10^6 cells with 1 microgram/mL of LPS and incubated for 24 hours (the same as what I did for the NO assay). According to the protocol in the kit, there is one step I need to add nuclear lysis buffer to the cell lysate and put in shake condition for an hour. After that, vortex vigorously. Hence, there are mechanical and chemical lysis happening here. Maybe I need to vortex longer to make sure that the nuclear envelope really breaks off? Or is it possible that the NF-kB signal is too low for detection?

So far I haven't try using TNF to activate the cells. But I'm trying to use H2O2+1 microgram/mL LPS to activate the cells (incubation hour: 24 hours). I'll run the assay again and I'll be getting the results this Friday.

Wish me best of luck. :)

One more question, is NO production linked with NF-kB activation? The pathways for both NO and NF-kB are different, right?

My work with NF-kB has shown that if you inhibit this transcription factor then NO levels can go down. There are studies that show that NF-kB activation can cause increased NO production.

Since you are looking at 24 h after stimulation, I would think your problem lies with the time point you've chosen. Generally speaking, p65 translocation to the nucleus is highest within an hour of stimlulation. After that p65 is ubiquitinated and degraded or shuttled back to the cytoplasm. While your system may be different, I quickly looked up the kit you're using and even the example experiment they provide with Jurkat stimulated with LPS shows the peak nuclear translocation is 30 min. I would do a course of 15, 30, 45 and 60 min stimulation to determine the maximum translocation. Maybe add a couple more time points if you have reason to believe the maximum translocation may occur later.

Thank you for your suggestions, Eric! I will try to run the assay again. Thank you so much. :)

Do you sure, that the ELISA KIT is especially for mouse!

Hi,

NFkB is a transcription factor activating the expression of the inducible NO synthase (iNOS). Therefore, increase of NO production reflects an increase of iNOS cellular activity and/or quantity. In other words, LPS-activated NFkB increases iNOS expression leading to an increase of NO production. This has been extensively shown and molecular mechanism regulating NFkB as well. You can check paper of Christopher K Glass from La Jolla University (USA) for this.

For your incubation with the nuclear lysis buffer, which must be an hypertonic buffer as usually used for this type of extraction, you may incubate your samples at 4ºC for 30 min while agitating (on a wheel for instance). You surely will improve the lysis efficiency.

As Eric and I said previously, if you are looking at NFkB activation, you need to check nuclear translocation between 10 min and 1 hour after activation. You can do a time point at 2 hour to be sure but it is a very quick phenomenon. As a functional test, you can do a luciferase assay as suggested above using pGL3-iNOSprom-Luc, ±siRNA p65/p50 or using the inhibitor JSH-23 (30-40 µM) to show the dependency of NFkB on the reporter gene response. Alternatively, you can check NFkB target genes by qPCR using the same conditions such as iNOS, IL1beta, NLRP3... As suggested above as well, you should consider WB on cytoplasmic and nuclear fraction, albeit a good cytoplasmic control is difficult to find. I would suggest Lamin A for the nucleus and calpain for cytoplasm. Good luck in your experiment.

Benoit

Thank you so much for your suggestions and comments, Benoit! They are very helpful.

Yes, I'm looking on NF-kB activation instead of quantifying the NF-kB protein. I'll do the time-course study and see whether I can succeed this time round.

Thank you again! :)

Hi Benoit. I couldn't find the paper written by Christopher K Glass regarding the iNOS and NF-kB. Do you mind share the link?

Thanks for your suggestion Aneesh! For immunofluorescence, I need to purchase specific antibody for p65 right? Is there any kit for this?

The antibody in this ELISA kit is provided separately.

Thank you. :)

an simple way to check for NFKb p65 activation is to do a Western on the nuclear lysates and see if you get an increase. I also use the AB from Santa Cruz. (#SC-372)

Hi,

You can check this paper, but this is not the group who has actually shown the regulation of iNOS by NFkB. This one is a nice paper with molecular regulation.

http://www.nature.com/nature/journal/v437/n7059/full/nature03988.html

The next oneis a nicework with other NFkB-dependent genes.

http://www.sciencedirect.com/science/article/pii/S1097276512006478

Enjoy the reading

What has the cell concentration been used?

Thank you so much, Benoit!

Hi,

One of the considerable thing is the lysate fraction,separation of Nuclear fractions and usage of hypo tonic buffers.The experimental conditions sounds correct and with LPS , after 15-30 min, the NFkB really at optimized activities.This is the stage for your cell crude and separated fraction preparation.

The easiest way to monitor is imaging the trans location of p65 towards nucleus by immuno florescence.

Check your protocol with this optimized reagents and tips.

http://txch.org/txch/assets/File/horton-nfkb-elisa-protocol.pdf

Good luck,

Hi!

Thanks for your help and suggestion, Aluru and Lucia~! :)

Only thing I can add is to make sure you have more than 5% serum in your media when you are treating cells with LPS. There are soluble factors in the serum that are required for TLR4 activation by LPS, such as CD14 and LBP.

I was able to strongly detect p-NFkB (Ser 468) with cat # 3039 and total NFkB with cat# 8242, both from Cell Signaling with only 19 ug of cellular lysates (not nuclear fraction).

Are you sure about cell concentration?

Hi!

I've incubated the macrophage cells using 1ug/mL of LPS for 24 hours. However, I didn't get any activation from that. I've consulted the technical person from IMGENEX (the company which I've bought the kit), he also mentioned that I need to do a time course to determine the optimum activation for the cells I'm working with.

According to the protocol in the kit, I have to harvest the cells after treatment and perform cell lysate immediately and store it in -80 degree celcius if I'm not using it immediately for NF-kB assay. But normally, I will stored it for a day after cell lysate because I need to coat the antibody and incubate it for 24 hours before putting in the sample.

About the cell concentration, I always do Bradford assay to determine the protein concentration before proceeding to NF-kB. Hence, I am sure that it is not the protein concentration that affects the activation of NF-kB.

Then you should start coating the plate the day before starting the cell treatment so it is ready to go when your cells are.

24h incubation is too long for NF-kB. Incubation for 2h and using the neuclear extraction ( about 10ug) will be no problem.

Did you try westren blotting of IkBa and/or P-IkBa? Degradation and phosphirylation of IkBa can be easily detected by using whole cell extracts.

You shoul better optimize incubation time and LPS concentration before NFkB experiment, in which much more cells are needed for neclear extract preparation than whole cell extarct.

24h maybe too long for detecting cytokines expression by ELISA. I did detect TNFa and IL-6, both were highest in about 6h and then they would decrease for unknown reason. So I suggest you to do a time course experiment to get the best point.

As many guys said. 24h is too long for detecting nfkb activation. Less than one hour should be better. Good luck!

@Pui-Mun Chan

Have you tried TNFa stimulation for approx. 20 min for control if your ELISA system works for your cells?

@Gabriele Froemming

Did you see a NF-kB activiatio after shorter incubation time with LPS. Because NFkB regulated factors can also activate NF-kB (e.g IL-8) and thus it might be a secondary NF-kB activation after 16 hours. A similar mechanism is discribed in Gangadharan et al. 2009 "Late phase activation of nuclear transcription factor kappaB by doxorubicin is mediated by interleukin-8 and induction of apoptosis via FasL".

Thanks everyone for your response! Appreciate it very much. :)

Currently I'm trying to find the optimum activation by doing a time course. Today, I've collected the nuclear fraction and did Bradford assay to check on the protein concentration. I've found that the protein concentration is about 1mg. Is it ok if I test the activation of NF-kB with that high of protein concentration? Or should I dilute it to 10ug instead?

The ELISA kit did not state the amount of protein to use. That's why I'm unsure whether I should dilute it or not ,or just use the stock concentration that I have. Btw, thanks for your comments, Vinod. :)

Pl try different dilutions of protein and choose the optimum dilution

lalitha kabilan

Hi!

I've done the following but still fail to get the NF-kB activation.

1) Incubated 1 million cells and 5 million cells with 1 ug/mL LPS for 15, 30, 45, 60, 75, 105, 120 minutes

2) Check for NO production to see whether my LPS is working or not. The LPS is definitely working fine.

Sigh. I think I will directly check the gene expression using RT-PCR.

Certainly NF-kB is actually being activated and translocated during that time. I would then guess the problem is you are not detecting the P65 (no signal) or you are getting your nuclear fraction contaminated with cytoplasm (equal signal for all samples) and so you are not detecting the translocation, since that kit detects all P65 (if I remember correctly).

Why don't you try to do an immunofluorescence or an immunocytochemistry to observe the translocation?