Figure 5. Protein release and IL-8 and TNF-α mRNA levels in primary human macrophages and response to LPS. 10⁶ primary macrophages from three healthy donors were seeded overnight, the following day the cells were washed and the medium was replaced, control cells were left untreated for 4 h (C 4 h]), or treated with 1 µg/ml LPS for 2 and 4 h. THP-1 cells were used as control and treated similarly. (A and B) Supernatants were collected and assayed for IL-8 and TNF-α using specific ELISA. (C and D) Total RNA was prepared from the cells and teal-time PCR was performed to quantify IL-8 and TNF-α mRNA levels. D: healthy human donor.