Bio plex mouse cytokine assay

To measure cytokines, inguinal lymph node cells or spleen cells were cultured (5 X 10⁵ CD4+ T cells/ml) with wild type APC’s (I-A^q-positive splenocytes) (1:2 ratio) which had been prepulsed with differing concentrations of the various peptides (A2, A9, or other variants). (Each cell population was confirmed by flow cytometry to have > 95% purity). Supernatants were collected at 72 hours and analyzed for the presence of IL-4, IL-10, IL-2, INFγ, and IL-17 using a Bio-plex mouse cytokine assay (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. Values are expressed as picograms per ml and represent the mean values for each group.

For infection with a hypervirulent strain of A. baumannii (HUMC1), 8- to 12-week-old age- and sex-matched mice were challenged intravenously via the tail vein with 1.2 × 10⁷ to 1.5 × 10⁷ colony-forming units. HUMC1 were grown overnight in tryptic soy broth at 37 °C with shaking. The bacteria were passaged to mid-log growth phase in tryptic soy broth at 37 °C with shaking. Cells were washed three times with PBS and resuspended at the appropriate concentration for infection. For survival studies, mice were monitored twice daily and were euthanized when they appeared moribund. Blood was obtained by retro-orbital bleeding or cardiac puncture. For quantifying bacterial burden, blood was serially diluted in PBS and plated on tryptic soy agar plates. Following overnight incubation at 37 °C, colony-forming units were counted. Plasma cytokine concentrations were quantified using a custom Bio-Plex mouse cytokine assay (Bio-Rad) and were analyzed on a Bio-Plex 200 plate reader using Bio-Plex Manager software 6.1 (Bio-Rad) at the USC Immune Monitoring Core.