Hi everyone,
I have trouble detecting IL-1b after human PBMCs were stimulated with LPS. If i mentioned about my experiment set:
1. 1x10^6 PBMCs/well were cultured at 24 well plate.
2. LPS at varying concentrations such as 30 ng/mL, 100 ng/mL and 1 ug/mL (final concentration) were added.
3. PBMCs were incubated for 6 h or 24 h.
4. Cell supernatant was collected to analyse IL-1b by ELISA.
In any case, i did not detect IL-1b by ELISA although the standards (IL-1b) were working well. Is there any suggestion and explanation for this problem?
Thank you for your help!
Dear Nilgun,
If you're not seeing anything by ELISA and your lab is set up for flow cytometry you could also look at IL1b or inflammasome activation that way. There are decent antibodies for full length pro-IL1b (for example eBioscience 12-7114 or Thermo Fischer 25-7114-80). For intracellular staining you'll want need to permeabilize the cells and may need to use GolgiPlug, GolgiStop, or brefeldin A. There are also some fluorescent caspase activity reporters (FAM-FLICA) that work for flow though these do not read out IL1b specifically.
cheers, Rob Hagan, UNC Chapel Hill
Hello Nilgun
I would like to draw your attention to the research article below. May be you will find an answer.
Article ATP-Induced IL-1β Specific Secretion: True Under Stringent Conditions
Regards.
Increase your cell concentration and reduce the volume of your cell culture. The signal is coming from the monocytes which represent only 20% or so of the PBMCs.
Alternatively, isolate and study monocytes using positive selection for CD14.
Hi Nilgün Yakuboğullari
IL-1b is produced in its inactive form after LPS stimulation. Mature IL-1b will be released by the cells after a second signal of stimulation. The article mentioned by Malcolm Nobre explains a little more about these mechanisms.
Also, try the suggestion of Caroline A Owen about increase cell density and reduce the volume of cell culture.
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