Fig 1: Detection of cytokines in the supernatant of RAW cells infected with cowpox virus, ectromelia virus and vaccinia virus. (A) From 2, 4, 6, 8, 10 and 16 h post-infection (hpi), the secreted cytokines were measured with the Bio-Plex Mouse Cytokine 23-Plex Panel (#M60009RDPD, Bio-Rad, Inc., Hercules, CA, USA). Data was analyzed using the Bio-Plex Manager software (Bio-Rad, Inc., Hercules, CA, USA) and pairwise analysis was performed with * representing p value < 0.05; ** < 0.01; and *** < 0.001. The amount of cytokines are indicated in pg/µL on Y-axis and time of infection as hpi is indicated on X-axis. (B) Table showing significant differences among the 3 viruses. C = cowpox virus, E = ectromelia virus and V = vaccinia virus. Up/Down indicates up-regulation or down-regulation of cytokine levels. Error bars are analysed from the average of 3 readings per sample.
Fig 2: Impaired the cytokine/chemokine induction by EPAC2 deficiency. (A–D) WT or EPAC2-/- mice were sham infected or infected with RSV as described in Figure 1 . The BAL fluid samples were collected at day two p.i., followed by cytokine/chemokines quantification using Bio-Plex Pro Mouse Cytokine 23-plex kit (Bio-rad, Cat #: M60009RDPD). The secretion is shown according to their absolute induction in the infected WT mice: 0-200 pg/ml (A), 400-1400 pg/ml (B), and more than 1500 pg/ml (C). (D) unaffected immune mediators. n = 12 mice/group. The results, shown as mean ± SE, are from three independent experiments. Asterisks indicate levels of significance, *P < 0.05 and **P < 0.01 for comparison to RSV-infected samples from WT mice. (E–G) Mice were treated with MAY0132 or vehicle, RSV at the dose of 3×106 pfu, or sham infected and harvested at day one postinfection to collect BALF samples to measure cytokines and chemokines by the multi-plex cytokine detection system. The mediators are shown in groups according to their absolute induction in the infected mice with vehicle treatment: 0-200 pg/ml (E), 400-1400 pg/ml (F), and more than 1500 pg/ml (G). The results, shown as mean ± SE (n = 6 mice/group). Asterisks indicate levels of significance, *P < 0.05 and **P < 0.01 for comparison to RSV-infected vehicle-treated mice. G-CSF, granulocyte colony-stimulating factor; KC, neutrophil chemokine; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; RANTES, regulated upon activation, normal T-cell expressed and secreted; TNF-α, tumor necrosis factor-α; IL, interleukin; IFN-γ, interferon-gamma.
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