Pe mouse anti human il 4

We conducted a coculture experiment with hUCB-MSCs and Th2 cells to assess inhibitory effect of hUCB-MSCs on Th2 cell differentiation. hUCB-MSCs (3 × 10⁴) were seeded on a Transwell membrane (pore size 8 μm; Corning, Bedford, MA, USA). CD4⁺ T cells were purified from human peripheral blood mononuclear cells (Lonza) using a human CD4⁺ T cell isolation kit (Miltenyi Biotec, Bisley, UK) according to the manufacturer’s instructions. After the attachment of hUCB-MSCs, 5 × 10⁵ CD4⁺ T cells were cultured in the well beneath the insert for 5 days. Cells were cultured with RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, Dynabeads Human T-Activator CD3/CD28 (Gibco BRL), IL-2 (20 ng/ml), IL-4 (20 ng/ml) and IL-6 (20 ng/ml) to induce Th2 differentiation. All recombinant human cytokines were purchased from Peprotech. For 5 days, CD4⁺ T cells were treated with pimecrolimus (100 ng/ml) at the indicated concentrations with or without hUCB-MSCs. After 5 days of coculture, Th2 differentiation was analyzed by detecting the levels of the CD4 (FITC mouse anti-human CD4; BD Biosciences) and IL-4 (PE mouse anti-human IL-4; BD Biosciences) proteins using a FACSCalibur flow cytometer and Cell Quest software (BD Biosciences).

PBMCs were analysed using flow cytometry (FACSVantage SE, BD, NJ, USA). Th1 and Th2 cells subsets were identified based on the detections of CD3 and CD4, as well as intracellular cytokines, IFN-c or IL-4. Briefly, cells were labelled with APC mouse anti-human CD3 and PE-Cy-™5 mouse anti-human CD8, and then stained with FITC-mouse anti-human IFN-c and PE-mouse anti-human IL-4 antibodies (all antibodies were from BD, NJ, USA). Th1 cells were labelled with CD3+ CD8- IFN-c+ and Th2 cells were labelled with CD3+ CD8- IL-4+. Number of cells was reported as a percentage of total CD3+ cells.