Figure 1.
Selenium supplementation promoted TCR and ConA-induced T-cell proliferation, but not unactivated and PHA-induced proliferation.
Primary porcine splenocytes (5×105 cells) were treated with different concentrations of sodium selenite for 48 h in the absence (A) or presence of anti-CD3 (B), ConA (C) or PHA (D). T-cell proliferation was analyzed using a WST-8 Cell Counting Kit-8. Cells without any stimulus were used as control (CON). Data represent mean ± S.E. of two independent experiments, each measured in quadruplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Figure 2.
CD3+ T cell proliferation monitored using CFSE labeling.
CFSE labeled splenocytes (2×106 cells/well) were treated with or without 2 µmol/L of sodium selenite in the absence or presence of anti-CD3, ConA or PHA, and proliferation was analyzed at 48 h of culture. Cells were stained with anti-CD3-PEcy5. CD3+ T cells from the entire well were analyzed for proliferation by flow cytometry. The percentage of proliferating cells for each culture is indicated. A representative experiment from two separate experiments is shown.
Figure 3.
IL-2 production in splenocytes treated with different concentrations of sodium selenite.
Primary porcine splenocytes were treated with different concentrations of sodium selenite for 48 h in the presence of anti-CD3 (A), ConA (B) or PHA (C). Cells without any stimulus were used as control (CON). Then the cell supernatants were collected and IL-2 concentration was determined by ELISA. Data represent mean ± S.E. of two independent experiments, each measured in triplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Figure 4.
Effects of sodium selenite on GPx1 mRNA levels in porcine splenocytes.
Primary porcine splenocytes (2×106 cells) were treated with different concentrations of sodium selenite for 48 h in the absence (A) or presence of anti-CD3 (B), ConA (C) or PHA (D). Cells without any stimulus were used as control (CON). GPx1 mRNA was measured by quantitative real-time RT-PCR, and the ratio of the level of GPx1 mRNA to that of the β-actin internal control was used for statistical comparison. Data represent mean ± S.E. of two independent experiments, each measured in triplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Figure 5.
Effects of Se supplementation on TR1 mRNA level in porcine splenocytes.
Primary porcine splenocytes (2×106 cells) were treated with different concentrations of sodium selenite for 48 h in the absence (A) or presence of anti-CD3 (B), ConA (C) or PHA (D). Cells without any stimulus were used as control (CON). TR1 mRNA was measured by quantitative real-time RT-PCR, and the ratio of the level of TR1 mRNA to that of the β-actin internal control was used for statistical comparison. Data represent mean ± S.E. of two independent experiments, each measured in triplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Figure 6.
GPx1 activity in splenocytes treated with different concentrations of sodium selenite. Primary porcine splenocytes were treated with different concentrations of sodium selenite for 48 h in the absence (A) or presence of anti-CD3 (B), ConA (C) or PHA (D).
Cells without any stimulus were used as control (CON). The activity of GPx1 in splenocyte cytosol was measured spectrophotometrically and expressed as units per gram of protein. Data represent mean ± S.E. of two independent experiments, each measured in triplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Figure 7.
GSH levels in splenocytes treated with different concentrations of sodium selenite.
Primary porcine splenocytes were treated with different concentrations of sodium selenite for 48 h in the absence (A) or presence of anti-CD3 (B), ConA (C) or PHA (D). Cells without any stimulus were used as control (CON). Total GSH concentration in splenocyte cytosol was measured spectrophotometrically. Data represent mean ± S.E. of two independent experiments, each measured in triplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Figure 8.
Effects of N-acetylcysteine (NAC) on mitogen-induced proliferation (A) and IL-2 production (B) of porcine splenocytes.
Primary porcine splenocytes were either unstimulated (None) or stimulated as indicated with anti-CD3, ConA or PHA in the absence or presence of 5 mmol/L of NAC. Data represent mean ± S.E. of two independent experiments, each measured in quadruplicate. Mean values without common letters within a given mitogen were significantly different (P<0.05).
Table 1.
Primers used for quantitative real-time PCR.