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. 2017 Feb 15;26(4):249–262. doi: 10.1089/scd.2016.0199

FIG. 2.

FIG. 2.

Activation of mesenchymal stem cells (MSC) with interferon gamma (IFNγ) enhances suppression of DC activation. DC were cocultured with MSC for 24–36 h that were either untreated or pretreated with 100 ng/mL IFNγ for 18 h. (A) Representative raw data of dot plots illustrating the differential expression of canine MHCII and CD86 between immature DC (iDC), lipopolysaccharide (LPS)-treated DC, LPS-treated DC with untreated MSC, and LPS-treated DC with IFNγ-conditioned MSC. (B) DC, alone or in coculture, with either untreated or IFNγ-treated MSC at ratios of 1:10, 1:100, or 1:1,000 (MSC:DC) were subsequently treated with LPS. DC were harvested by gently removing nonadherent (or loosely adherent) cells from MSC and immunostained with mouse anti-canine CD11c (followed by either donkey anti-mouse-conjugated APC or -Alexa Fluor® 488) and either FITC-conjugated anti-MHCII and PE-conjugated anti-CD86 or APC-conjugated anti-CD40. Cytometric analysis was done by gating on large cells expressing high levels of CD11c. Data are from triplicate cultures and are representative of DC cultures established from seven different dogs. (C) DC were incubated at a ratio of 1:10 MSC:DC for 24–36 hr with MSC that were untreated or IFNγ-treated (100 ng/mL), followed by DC activation with LPS (50 ng/mL). Supernatants from DC alone and MSC:DC cocultures were analyzed for canine tumor necrosis factor alpha (TNFα) and interleukin-10 (IL-10) concentrations using canine specific ELISAs, according to the manufacturers' protocols. In addition, supernatants from similar numbers of MSC alone either treated with LPS and/or IFNγ were analyzed for TNFα/IL-10 secretion. Data represent TNFα secretion (i) and IL-10 secretion (ii). Data are representative of four independent experiments (four different dogs). Values are expressed as geometric mean fluorescence intensity (MFI). P values were calculated for statistical variance using a paired two-tailed Mann–Whitney test. (“ns”, not significant; *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001).