I am doing the cytokine expression experiment. I used the PMA (5ng/ml) to stimulate the THP-1(1x106 cells/ml) in the flask. After 48h, I replaced the PMA medium with serum free RPMI1640 medium (no FBS) and maintained in the incubator for 3hr. Then, the 100ng/ml LPS was added for cytokine stimulation, and I collected the supernatant at 12h and 24h. But the human inflammatory cytokine kit cannot detect another 5 kinds of cytokine including IL-1b, TNF, IL-10, IL-6 and IL-12p70 (expect for IL-8), so I want to ask if there are some mistakes in my experiments? Or the cytokine levels are below the detection range of cytokine kit (20pg/ml)? Hope you can help me to answer this question, Thanks a lot!!
There are so many factors that can affect the results of your experiments. Some of the parameters that I can think of are:
1) Contamination like mycoplasma can affect the results of the cytokine detection. https://www.ncbi.nlm.nih.gov/pubmed/20360862
2) Concentration of LPS (In my experience, I used higher dose which was 1 µg/ml and I could detect IL-1b, IL-6 and TNF-a in my cells, but not IL-10). Same parameters for my colorectal cell line, IL-8 was detected.
3) Effect of culture conditions of the THP-1 cells. It is crucial to maintain the same density for every experiment, constant counting and monitoring, and also preferably higher density for the THP-1 cells in the flask. Check this: https://www.ncbi.nlm.nih.gov/pubmed/23621670
4) Another thing that I observed is you might want to leave your PMA-treated THP-1 cells in resting phase for longer period (3 to 5 days, instead of 3 hours). Check this: https://www.ncbi.nlm.nih.gov/pubmed/20084270
Collection supernatant at 12 h and 24 h sounds fine to me, in my experience.
Dear Liu, I suggest you to reduce the PMA stimulation to 5h followed by allowing cells to attain 90-100% confluency (48-72h). LPS (50-100ng/ml) for 5-6h may be enough (you may need to optimize it) for getting required expression, instead of upto 24h. You may also target expression studies at transcription level.