Fig 1.
Porcine MoDCs at different culture times (400×).
CD14+ monocytes were purified from porcine PBMCs and were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 25 ng/mL recombinant porcine IL-4, and 10 ng/ml recombinant porcine GM-CSF. The culture medium was replenished every 3 days. (A) CD14+ monocytes after 2 days of culture. (B) CD14+ monocytes 3 days of culture. (C) CD14+ monocytes after 4 days of culture. (D) CD14+ monocytes after 5 days of culture.
Fig 2.
Identification of 5-day old MoDCs by FCM.
106 of 5-day old MoDC cells were harvested and stained with mouse anti pig CD1, mouse anti pig SLA-II, mouse anti porcine CD172a and analyzed on a FACSCalibur flow cytometer (BD Biosciences, Hiedelberg, Germany) by using CellQuestPro software (BD Biosciences).
Fig 3.
Expression levels of major surface markers on 7-day old MoDC.
6-day old MoDCs were treated with LPS (1 μg/mL), Pam3CSK4 (500 ng/mL) or SC19 (MOI = 0.1) for 24 h, and expression levels of major surface markers on MoDC were analyzed by flow cytometry. (A) Three different panels indicating the CD1, CD172a and SLA-II expression. (B) The representative dot-plot of flow cytometry analysis data for the CD1, CD172a and SLA-II expression.
Fig 4.
Phagocytic ability of MoDCs treated with neutral red.
(A) OD570nm of MoDCs at different culture times. (B) OD570nm of 8-day old MoDCs when stimulated with SC19 (MOI = 0.1), LPS (1 μg/mL) or Pam3CSK4 (500 ng/mL) for 2 days.
Fig 5.
SI of T cells stimulated by MoDCs.
5-day old MoDCs were treated with SC19 (MOI = 0.001), LPS (1 μg/mL) or Pam3CSK4 (500 ng/mL) for 24 h, and then co-cultured with T cells in different ratios. Three days later, OD450nm of each well was detected by microplate reader and SI was calculated.
Fig 6.
Cytokines secreted by MoDCs in response to the SC19 stimulation.
5-day old MoDCs were stimulated by SC19 (MOI: 0.1, 0.01, 0.001), LPS (1μg/mL) or Pam3CSK4 (500ng/mL). After 12 and 24 h, supernatants were collected and analyzed for cytokines using Porcine Cytokine Array.
Fig 7.
Purity of CD4+ T cells analyzed by flow cytometry.
Porcine peripheral blood CD4+T cells were purified from PBMCs by immunomagnetic labeling of cells using mouse anti-pig CD4a monoclonal antibody and anti-mouse IgG MicroBeads. Cells were stained with PE Mouse Anti-Pig CD4a. Flow cytometry was performed using a FACSCalibur to analyze the purity of CD4+T cells.
Fig 8.
Differentiation of CD4+ T cells co-cultured with MoDCs and SC19.
(A) The proportion of Th-1 cells. (B) The proportion of Th-2 cells. CD4+T cells were mixed with 5-day old MoDCs in the proportion of 4:1, both of them were stimulated by SC19 (MOI = 0.1); after 12 h, cells were analyzed by flow cytometry.
Fig 9.
Cytokine in the supernatants of CD4+ T cells and MoDCs stimulated by SC19.
CD4+ T cells were mixed with 5-day old MoDCs in the proportion of 4:1. The mixed cells were stimulated by SC19 (MOI = 0.1), LPS (1 μg/mL) or Pam3CSK4 (500 ng/mL) at 37°C with 5% CO2. 17 hours later, supernatant from each well were collected and analyzed for IL-4, IL-17, IL-12p70, and IFN-γ using Human Th1/Th2/Th17 Antibody Assay.