Human umbilical cord MSCs (UC-MSCs) were isolated in our laboratory by processing human umbilical cord tissue as described by Professor Hou et al. [22 (link)]. Then, the cells were routinely resuspended in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, USA) supplemented with 10 % fetal bovine serum (Hyclone Laboratories, USA) and 100 U/ml penicillin/streptomycin (Gibco Life Technologies, USA). The cultures were maintained at 37 °C in 5 % CO2 and 95 % humidity, and cells at the 4th–5th passages were used for the subsequent experiments.
For LPS preconditioning, 1.5 × 106 UC-MSCs per 15-cm cell culture dish were seeded for 24 h to achieve a confluence of 70–80 %. After the medium was aspirated, the cells were rinsed three times with PBS and treated with LPS (100 ng/ml in serum-free medium, Sigma, USA) or serum-free medium alone as a negative control and then incubated for 2 days prior to supernatant collection.
For LPS preconditioning, 1.5 × 106 UC-MSCs per 15-cm cell culture dish were seeded for 24 h to achieve a confluence of 70–80 %. After the medium was aspirated, the cells were rinsed three times with PBS and treated with LPS (100 ng/ml in serum-free medium, Sigma, USA) or serum-free medium alone as a negative control and then incubated for 2 days prior to supernatant collection.
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