Dexamethasone (dex)

Eosinophils from murine intestines were cultured at 37℃ and 5% CO₂ in RPMI containing 1% penicillin/streptomycin and 10% FBS in 96-well at a cell concentration of 2.0×10⁴ cells/well. Eosinophils were cultured in the presence of recombinant interleukin 5 (IL-5) (1 ng/mL; R&D Systems, Minneapolis, MN, USA) and dexamethasone concentration (0 to 100 nM), and survival rate in the presence of dexamethasone was compared to viability in recombinant IL-5 medium alone. Viable cells were assessed by the trypan blue exclusion method. Cell viability was observed at day 3 for all experiments and was calculated by the total cell count minus the count of nonviable or dead cells.

To induce healthy and CP patient MSC to differentiate into various cell phenotypes, cells (0.5 × 10³ cells per cm²) were plated in 12‐well culture plates and allowed to reach confluence. Osteogenic differentiation medium, consisting of complete culture medium supplemented with 50 μg/ml ascorbic acid, 10 mM β‐glycerolphosphate, and 10 nM dexamethasone (all from Sigma), was exchanged every 3 days for 3 weeks. The cells were fixed with 10% neutral buffered formalin for 30 minutes, and then stained with Alizarin Red for 1 hour. For adipocytes differentiation, complete culture medium was supplemented with 50 μg/ml ascorbic acid, 0.5 mM 1‐methyl‐3‐isobutylxanthine, 10 nM dexamethasone, and 10 μg/ml insulin. The cells were fixed with 10% neutral buffered formalin for 30 minutes, and then stained with Oil Red O for 50 minutes. Chondrogenic differentiation was induced using a pellet culture system by culturing the cells pellet in 15‐ml conical tubes with media containing DMEM high glucose, 5% FBS, 1% l‐glutamine, supplemented with 10% ITS+ Premix tissue culture supplement (Invitrogen, Carlsbad, CA), 100 nM dexamethasone and 10 ng/ml TGF‐β (R&D system, NE Minneapolis, MN) for 21 days with medium exchange twice a week. The aggregates were fixed with 10% neutral buffered formalin overnight, frozen in O.C.T and sectioned. Alcian blue was used to stain the extracellular matrix.

For chondrogenic differentiation, cells were cultured in a three-dimensional pellet culture: 200,000 cells were suspended in 0.2 mL of chondrogenic-inducing differentiation medium, consisting of DMEM high glucose, 1% penicillin/streptomycin, 1% ITS+ premix (354352; BD), 0.04 mg/mL proline (Sigma), 0.1 mM ascorbic acid, 0.1 μM dexamethasone, and 10 ng/mL TGF-β1 (240-B-002; R&D Systems). The pellets of the control group were suspended at the same density in 0.2 mL of chondrogenic differentiation medium without TGF-β1. Initially, L-MSC chondrogenesis was not induced; therefore, in a follow-up experiment, we cultured L-MSC pellets in the presence of TGF-β1 (10 ng/mL) and BMP-2 (250 ng/mL; R&D). The suspensions were placed in a 96-well round-bottom polystyrene plate (Corning Costar 7007) resulting in ultralow attachment of the cells, which was centrifuged for 5 min at 1,500 rpm at RT. Medium was changed every day for 2 weeks and thereafter every other day for another week. After 21 days, three pellets were collected from every condition for RNA isolation and qPCR analysis and two pellets for histological evaluation. We stained 5 μm thick sections with 0.125% Safranin O (Sigma) for proteoglycans in cartilage and counterstained with 0.4% Fast Green (Sigma); differentiation status was assessed by staining and morphology of the cell pellet.