Cell proliferation elisa bromodeoxyuridine brdu kit

The Cell Proliferation ELISA bromodeoxyuridine (BrdU) Kit used for this assay was purchased from Roche (Basel, Switzerland). Cells were seeded (0.5 × 10⁵ cells/well) and incubated at 37 °C in a 5% CO₂-humidified atmosphere for 24 h. After 24 h, the cells were labeled with 10 µL/well BrdU labeling solution, and then re-incubated for an additional 4 h at 37 °C in a 5% CO₂-humidified atmosphere. After removing the media, Fix Denat solution was added to each well, incubated at room temperature (RT) for 30 min, and then removed. An anti-BrdU-peroxidase working solution was added to each well and incubated for a further 90 min at RT. The cells were then washed with washing solution three times, and 100 µL of substrate solution was added to each well and incubated for 30 min. Cell proliferation was estimated by measuring absorbance at 370 nm.

PBMCs were stimulated with 10 μg/mL PHA and seeded at 20 × 10⁴ cells in a 96‐well plate in the presence or absence of 20 × 10³ mitomycin‐C (25 μg/mL) treated AFSCs, BM‐MSCs, or PLSCs. Stromal cells were plated into a 96‐well plate and allowed to adhere overnight. The coculture system was incubated for 6 days before cell proliferation was assessed using the Cell proliferation ELISA bromodeoxyuridine (BrDU) kit (Roche, Basel, Switzerland). An equivalent of 0.1 mM BrDU was added on day 5 for an overnight incubation.

TMSCs with or without cytokine priming were treated with 25 mg/mL of mitomycin C (Sigma-Aldrich) at 37 °C for 1 h to hinder cell proliferation, followed by seeding into 96-well plates at a density of 1x10⁴ cells/well. Peripheral blood mononuclear cells (PBMCs, Zenbio, Research Triangle Park, NC, USA) were added to TMSCs-plated well for coculture in RPMI1640 media (Gibco) containing 10% FBS in the presence of concanavalin A (ConA 5 µg/mL, Sigma-Aldrich) or anti-CD3 (5 µg/mL)/anti-CD28 (2 µg/mL, eBioscience, San Diego, CA, USA) for the activation of pan-leukocytes or T lymphocytes, respectively. The proliferation of PBMCs or T lymphocytes was determined using Cell Proliferation ELISA, bromodeoxyuridine (BrdU) Kit (Roche, Indianapolis, IN, USA) following 5 days of coculture.
To assess the immunogenicity of TMSCs, naïve and primed TMSCs were cocultured with the PBMCs (TMSCs:PBMCs = 1:10) without any stimuli, and the PBMC proliferation was measured compared with the results from PBMCs treated with mitogen or immune stimulants such as ConA and anti-CD3/28. To evaluate the immunosuppressive effects, PBMCs were added to TMSCs at the ratio of 1:10 (TMSCs:PBMCs) under stimulation by ConA or anti-CD3/28 plus IL-2 (Peprotech). After 5 days of coculture, cell proliferation was measured by BrdU-incorporated colorimetric assay.