Fig. 3
CsA enhances IDO production and activity by IFNγ activated MSC. MSCs were seeded at 2 × 105 per well in a 6 well plate and left alone or stimulated with IFNγ (50 ng/ml) and/or CsA (1000 ng/ml) at different timepoints. In some groups, MSCs were first stimulated with IFNγ for 24 h followed by CsA for a further 24 h. After a total of 48 h, cells were harvested for Quantitative PCR (qPCR) analysis. In other groups, MSCs were stimulated with IFNγ and CsA and harvested after 24 h. Controls for exposure of cytokines at both 24 h and 48 h timepoints were performed. RNA from each sample was extracted, cDNA was synthesised and used as a template for qPCR. mRNA expression of IDO (a) was calculated relative to the house keeping gene GAPDH. The concentration of kynurenine was examined by colorimetric assay using a L-kynurenine standard curve to calculate kynurenine concentration (d). These experiments were repeated using four MSC donors (n = 4) (passage 5–7) and the results shown are representative of this. Statistical analysis was carried out using unpaired Student’s t test where * < 0.05, ** < 0.01 and *** < 0.001. Stars with no bar are in comparison to the MSC alone group