Hi,  when you want to detect intracellular cytokines/secreted cytokines, the day before your experiment you should plate the cells in cytokine free medium. (I see that you have added TGFb into the medium. TGFb might interfere with the DC activation by LPS)

Secondly, LPS is used mostly at a standard concentration of 1microgram/ml. But you have used 100 gram/ml which I think might be an extremely high concentration which would drive the cell into exhaustion.

Please use cytokine free medium and 1microgram/ml LPS and I hope that the problem is resolved.

Best!

Hi,

I have few suggestions for you-

1. For detection of intracellular cytokines using LPS as positive control, you should use lower concentration of LPS ranging from 10 ng/ml-1 microgram/ml but NEVER more than that. Because overdose of LPS can cause TOLERANCE.

2. During Bref A treatment to block cytokine secretion, we usually, incubate for 4-5 hrs but not more than that. I saw that you have incubated your samples with Bref-A for 16 hrs, that might be toxic to cells also.

3. What cytofix/cytoperm buffer you are using? I can suggest you to use the following buffer for fixation/perm- (2% paraformaldehyde + 0.05% saponin + 3% FBS in PBS) for 20 minutes at RT and for perm wash buffer-(0.05% saponin + 3% FBS in PBS)

Hope this will work.

Hello Anchana,

Thank you very much for your help.

Actually I wrote the LPS concentration wrong, it is not 100g/ml it is 100 ng/mL.

The question about the cytokine free medium I thing it's quite difficult to have, because I'm using DC differentiated from monocytes, so I have to add in the medium IL-4 and GM-CSF. However, as I'm trying to establish a tolerogenic DC, I added TGFb and IL-10.

Hello Debanjan, 

Thank you very much for your suggestions.

1 - I used 100 ng/mL because it is the same concentration I used to stimulate the DC to see phenotype and proliferation. I did not known that for ICS lower doses of LPS would be better.

2 - Yes, I have seen the majority of protocols that used Bref A for about 5-6 hours, but as the detection of IL-10 is very difficult I was afraid that I could lose cytokine produced in the first hours of activation, and thought that, even if I lost some cells due to Bref A cytotoxicity, I could have better cytokine detection.

3 - The cytokine/cytoperm buffer I'm using is the BD Cytophix/Cytoperm Buffer. I know the fixation/perm buffer has 4,2% paraformaldehyde and I'm staining the cells according to BD procedure.