Macsquant software

Expression of adhesion molecules on monocyte subsets was measured directly ex vivo by flow cytometry (MACs Quant 10; Miltenyi Biotec, Bergisch Gladbach, Germany). Fresh blood samples were lysed with FACS lyse buffer (BD Biosciences) for 15 minutes and washed with buffer (phosphate-buffered saline with 1% bovine serum albumin and 0.1% sodium azide). Cells were stained for 30 minutes in the dark on ice and then washed in buffer, fixed in 1% paraformaldehyde, and analyzed. Monocyte subsets were identified by size, granularity, and surface expression levels of CD14 and CD16 using fluorochrome labeled antibodies (anti-CD14 Pacific Blue and anti-CD16 phycoerythrin; BD Pharmingen, San Diego, CA). Fluorescence minus 1 and isotype gating strategies were used to identify expression of surface markers as previously reported [22 (link)].
Adhesion molecule expression was monitored using fluorochrome-labeled antibodies against: CD11a (Pe-Cy7), CD11b (allophycocyanin-Cy7 [APC-Cy7]), CD11c (APC), CD18 (fluorescein isothiocyanate [FITC]), CD29 (APC), and CD49d (Pe-Cy7) (BD Pharmingen). Surface expression of CX3CR1 was measured using anti-CX3CR1 (Pe-Cy7; eBioscience, San Diego, CA). MACS Quant software (version 2.21031.1; Miltenyi Biotec) and Prism 5.0 GraphPad software (GraphPad, La Jolla, CA) were used to analyze the data.

Antigen-specific cell proliferative responses were measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Two weeks after the booster immunization, PBMCs were harvested by histopaque density centrifugation (Sigma, Germany). Cells were seeded in 96-well plates (1 × 10⁵ cells/well), stimulated with purified Salmonella outer membrane proteins (Omps) at 400 ng/well concentration, and incubated for 72 h at 37°C in a 5% CO₂ atmosphere. After incubation, cells were treated with MTT solution for 4 h, and the level of formazan production was measured at 570 nm. Further, to analyze T-cell responses following immunization, 1 × 10⁵ cells were seeded in 96 well plates and stimulated with Omps (400 ng/well) for 72 h. Following incubation, cells were labeled with anti-chicken CD3a-FITC (Cat # 8200-02), anti-chicken CD4-AF-700 (Cat # 8210-27), and anti-chicken CD8-PE (Cat # 8220-09) antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min. The T cell population was quantified using MacsQuant software (Miltenyi Biotec, Bergisch Gladbach, Germany).