Magnetic activated cell sorter immunomagnetic separation system

Single-cell suspensions from ESC-derived cells were stained with PE labelled
anti-huEpCAM antibody, washed, and stained with anti-PE MicroBeads (Miltenyi
Biotec, Auburn, CA). EpCAM⁺ and EpCAM⁻cells were selected using a magnetic-activated cell sorter immunomagnetic
separation system (Miltenyi Biotec).

Splenocytes (normalized to 1 × 10⁵ T cells per well) from HSCT recipients were cultured in the presence or absence of irradiated (2,000 cGy) splenocytes (2 × 10⁵ cells per well) from different mouse strains in a 96‐well plate for 4 days. Cell proliferation was measured by BrdU Labeling and Detection Kit III (Roche Applied Science, Mannheim, Germany,
https://lifescience.roche.com) according to the manufacturer's instructions. Absorbance (measured as optical density [OD]), proportional to BrdU uptake, was measured at 405 nm using an ELISA microplate reader (BioTek, Winooski, VT,
http://www.biotek.com). The data are expressed as stimulation index (OD in mixed leukocyte reactions [MLRs]/OD in spontaneous proliferation).
For one‐way MLRs, splenocytes from HSCT recipients were depleted for CD25⁺ T cells before MLR. Briefly, splenocytes were incubated with anti‐CD25‐PE, and then anti‐PE‐labeled microbeads (Miltenyi Biotec). Depletion of CD25⁺ cells was achieved by using a magnetic‐activated cell sorter immunomagnetic separation system (Miltenyi Biotec) as described 29. The purity of the depletion using this procedure, assessed by flow cytometry, was >97%.