An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

To perform an effector suppression assay, begin by setting up an MSC-PBMC co-culture with 2.5 x 10⁶ PBMCs, co-cultured with 250,000 MSCs to ensure enough MSC co-cultured PBMCs for subpopulation selection.

After 48 to 72 hours at 37 degrees Celsius, select the MSC-induced immunomodulatory leukocytes of interest with the appropriate magnetic beads. Then, add CFSE-labeled allogeneic CD4-positive T cells in 1 milliliter of complete leukocyte medium per well, at various experimental ratios.

Next, add anti-CD3/28-conjugated microbeads at a 1-to-1 bead-to-cell ratio to stimulate the CD4-positive T cells. On the third day of co-culture, assess the proliferation of the CFSE-labeled CD4-positive T cells by flow cytometry.