Imiquimod

All cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere. Medium composition for primary culture is described below. RAW 264.7 cells and HEK293T cells were grown in DMEM (Wako Chem., Osaka, Japan) supplemented with 10% FCS (Sigma-Aldrich, St Louis, MO), 100 U/ml penicillin, and 100 U/ml streptomycin (Wako Chem.). The TLR ligands poly I:C (Imgenex, San Diego, CA), LPS (Sigma-Aldrich), imiquimod (Imgenex), CpG-A (1585: Nihon Gene Research Laboratories, Sendai, Japan) and CpG-B (1668: Nihon Gene Research Laboratories) were used for TLR stimulation. N,N,N’,N’-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN; Sigma-Aldrich) was used for zinc chelation, and zinc loading of cells was performed by the addition of zinc sulfate (Sigma-Aldrich) and the ionophore pyrithione (Sigma-Aldrich). Doxycycline hydrochloride (Wako Chem.) was used at 1 μg/ml for the pTet-off system.

The method to isolate MSCs from umibilical cord was based on published literature [28 (link)]. The umbilical cord was diced into pieces after disinfection in 75% ethanol for 10–20 min. The small pieces of mesenchymal tissues were put into 25 mm² plates. The culture medium was added when tissues sticked tightly to the bottom of plates. The dissociated MSCs were collected about 5–8 days after the mesenchymal tissues were removed. The UCMSCs were then maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin and then stored in liquid for future used.
TLR7 agonist Imiquimod was purchased from IMGENEX (IMG-2207) and dissolved in DMSO at a concentration of 2.5 mg/ml. UCMSCs were seeded into 6-well plate at a concentration of 1.5 × 10⁵ in 2 ml medium and Imiquimod was added into the medium at final concentration of 10 μg/ml.