Skip to main content
. 2015 Jul 15;10(7):e0133385. doi: 10.1371/journal.pone.0133385

Fig 5. 4T1-conditioned macrophages demonstrate increased nitrite production in response to LPS.

Fig 5

BMDMs were treated with 4T1- or HC11-conditioned medium for 24 h. (A) To assess nitrite production, BMDMs were then washed with PBS followed by stimulation with LPS (100 ng/ml), flagellin (5 μg/ml), peptidoglycan (10 μg/ml), or CpG ODN (5 μg/ml) for another 24 h. Culture supernatants were collected and the nitrite production was assessed by the addition of Griess reagent for 15 min. Absorbance (570 nm) of the culture supernatants was then measured by a spectrophotometer. (B) To measure the production of reactive oxygen species, tumor-conditioned BMDMs were stimulated with bacterial TLR agonists for 24 h in the presence of Amplex Red reagent and horseradish peroxidase. Absorbance (450 nm) of the cultures was then measured by a spectrophotometer. Results are shown as the mean (± SEM) of at least 4 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; ** p < 0.01, *** p < 0.001.