Cd56 magnetic beads

The effect of SAHA-treated MSC on NK cell function was assessed by BATDA cytotoxicity assays as described previously [34 (link)]. MSCs were seeded into a 24-well plate and treated with 0.1–3.5 μM SAHA or corresponding controls for 48 h. NK cells were isolated by immunomagnetic selection using CD56⁺ magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) from PBMC of healthy donors. Prior to co-cultivation, SAHA-treated MSCs were washed with PBS (Biochrom, Berlin, Germany). NK cells were co-cultured with MSC at a ratio of 4:1 in the presence of 100 U/mL IL-2 (BD Biosciences, Heidelberg, Germany) where indicated. After four days of co-culture, NK cells were isolated and counted in a Neubauer chamber. The leukemic cell line K562 (ATCC) was used as target cell line. K562 was loaded with BATDA according to the manufacturer`s protocol (DELFIA Cell Cytotoxicity kit, PerkinElmer LAS, Rodgau-Jügesheim, Germany). NK cell cytotoxicity against the leukemic cell line K562 (ATCC, Manassas, VA, USA) was tested in triplicates. Effector cells and target cells were incubated for 2 h. Maximum lysis was reached using DELFIA Lysis buffer (PerkinElmer LAS, Rodgau-Jügesheim, Germany). Specific lysis was calculated using the following formula: $$\%\mathrm{Specific}\mathrm{lysis}=\frac{\mathrm{Lysis}\mathrm{by}\mathrm{effector}\mathrm{cells}-\mathrm{spontaneous}\mathrm{lysis}}{\mathrm{Maximum}\mathrm{lysis}-\mathrm{spontaneous}\mathrm{lysis}}\times 100$$