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!!
Hi Ya Liu, I'm thinking either the cells were not given enough time to recover after PMA differentiation (24h may be good) or/also, the cells were not differentiated before being stimulated with LPS. Ref:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545815/
