CD3/CD28 T cell proliferation assay

To evaluate the immunomodulatory capacity of MP, PBMC were labeled with 1 μM of CFSE and plated in round bottom 96-well culture plates at a density of 5 × 10⁴ cells/well. T cell proliferation was stimulated by adding human anti-CD3/anti-CD28 antibodies (1 µl/ml each) with a linker antibody Ig (2 µl/ml) (BD Biosciences). PBMC were incubated with different ratios of MP, or MPγ (1:5,000, 1:10,000, 1:40,000, 1:80,000) for 4 days. On the fourth day, non-adherent PBMC were removed from the plate, washed with FACS Flow and incubated with monoclonal antibodies against CD4-PerCP and CD8-PE-Cy7 (antibodies were purchased from BD Biosciences) at room temperature for 30 min. When a CFSE-labeled cell divides, its progeny are endowed with half the number of CFSE-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence by flow cytometry.