Apc anti human cd56

The KMS11, OPM2, and KMS12 target cell lines were first cultured overnight with 1 µM DAPT, stained with 0.5 µM 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma-Aldrich, Merck KGaA), and finally co-cultured with effector PBMCs, at a 5:1 E:T ratio, in the presence or absence of an equimolar concentration of BCMA×PDL1 bsAb or mAbs. DARA was used as positive controls. After 24 h [19 (link)], cells were harvested and stained using the GFP-certified TM Apoptosis/Necrosis detection kit (Enzo Life Science, Farmingdale, NY, USA), according to the manufacturer’s instructions. Samples were analyzed by flow cytometry (FACSCanto II instrument). Cell mortality is expressed as % Cytotoxicity calculated with the formula: $$\% \mathrm{Cytotoxicity}=\frac{\mathrm{\%} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{h} {\mathrm{C}\mathrm{F}\mathrm{S}\mathrm{E}}^{+}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}- \mathrm{\%} \mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{o}\mathrm{u}\mathrm{s} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{h} {\mathrm{o}\mathrm{f} \mathrm{C}\mathrm{F}\mathrm{S}\mathrm{E}}^{+}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{100-\mathrm{\%} \mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{o}\mathrm{u}\mathrm{s} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{h} {\mathrm{o}\mathrm{f} \mathrm{C}\mathrm{F}\mathrm{S}\mathrm{E}}^{+}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times 100$$
In some cases, NK cell activation was measured after 4 h of co-culture of PBMC and KMS11 cells at a 5:1 E:T ratio, by staining with anti-human CD56-APC (BD Biosciences) and anti-human CD107a-PE (BD Biosciences) mAbs [14 (link)]. The degranulation of NK cells was quantified by flow cytometry (FACSCanto II instrument) as an increase in percentage of CD107a⁺ cells in the CD56⁺ population.

Isolated muscle precursor cells were propagated in growth medium as described above until 70% confluence. Cells were detached using TrypLE^TM Express, and subsequently washed twice in FACS buffer [phosphate-buffered saline (PBS) containing 2% heat inactivated FBS]. Cells were stained with anti-human CD56-APC, CD31-PE, and CD45-BV421 (all from BD Bioscience) for 20 min and subsequently washed twice in FACS buffer. Data was acquired using a FACS Fortessa (BD Biosciences). For compensation, single stain was used with one drop of negative control beads and anti-mouse IgG beads (BD Biosciences). Data analysis was performed using FlowJo software version 10.