Human nk cell isolation kit

NK cells ready for transfection, as well as following flow cytometry and RT‐qPCR were purified from healthy volunteers‐derived PBMCs (n = 3) and MM patients‐derived PBMC (n = 3), according to the manufacture's instruction of the Human NK cell Isolation Kit (Miltenyi Biotec, Cologne, Germany). Then, the cells were fluorescently stained with PE anti‐human CD56 antibody (Biolegend, California, USA) and FITC anti‐human CD3 antibody (Biolegend) and analysed by flow cytometry to guarantee a purity of more than 95% CD3‐CD56 + NK cells. Expansion procedures have been described by Wagner et al.³¹ Briefly, K562‐based artificial antigen‐presenting cells expressing membrane‐bound interleukin (IL)‐21 (K562‐mb21‐41BBL feeder cells) were used to expand NK cells. They were irradiated at 100 Gy and added at a feeder cell: PB‐NK ratio of 10:1. NK cells (1 × 10⁶) were cultured in SCGM medium (CellGenix, Portsmouth, NH) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA), 400 U/ml recombinant human IL‐2 (R&D Systems, Minneapolis, MN), and K562 cells at 1 × 10⁷. During the 7‐day culture, IL‐2 and freshly prepared K562 cells were replenished every 2 days.³²

Following thaw of PBMCs (day 0 NK cells) or after 12 days of coculture of bulk PBMCs with K562-mb15-4-1BBL, NK cells were isolated via CD14+ cell depletion with the use of a magnetic bead selection kit (STEMCELL Technologies), followed by NK cell enrichment using a magnetic bead negative selection kit (human NK cell isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Before functional studies, NK cells were rested in NK media for 24 h.

Peripheral blood mononuclear cells (PBMNCs) were isolated from 10 normal healthy subjects using a Ficoll-Paque Leucosep™ density gradient solution (Greiner Bio-One, Kremsmünster, Austria). NK cells were isolated from PBMNCs using a human NK Cell Isolation Kit (catalogue number 130–092–657, Miltenyi Biotec, Bergisch Gladbach, Germany) as instructed by the manufacturer. Briefly, PBMNCs were resuspended in PBS solution (pH 7.2) containing 2 mM EDTA and 0.5% bovine serum albumin, and then the cells were incubated with biotin-antibody cocktail at 4 °C for 5 min. Following incubation of cells with microbead cocktail at 4 °C for 10 min, the cells were washed and resuspended in PBS buffer solution, and then loaded onto a column positioned in a magnetic separator (MACs; Miltenyi Biotec). Cells were then allowed to pass through and the effluent containing the unlabeled enriched NK cells was collected. The viability and purity of NK cells was evaluated byTrypan blue and anti-CD56 monoclonal antibody (R&D Systems) in flow cytometry, respectively. The viability and purity of NK cells used in this study were more than 95%. After isolation, NK cells were used either immediately or after 72-h activation with 100 U/mL IL-2 (R&D Systems) in RPMI 1640 medium containing 10% FBS, 2 mM l-glutamine, 50 μg/mL streptomycin, and 50 U/mL penicillin (NK culture medium).

ADCC target cell line was a multiple myeloma‐derived suspension cell line with high antigen expression recognized by mAb2. The target cells were seeded at a density of 15,000 cells/well in a 96‐well U‐bottom plate in a 50uL volume of ADCC assay buffer (serum free RPMI‐1640 media +1%BSA + 2 mM L‐Glutamine). Cells were then incubated with serial dilutions of test antibodies for 30 min at 37°C in 50uL of ADCC buffer. Target cells were finally exposed to purified NK cells in 50 ul ADCC buffer at a ratio of 3:1 (effector: target) to induce ADCC mediated cell lysis. The ADCC lysis endpoint was measured after 4 hr in culture at 37°C by collecting 100 ul of supernatant from each well. Cell lysis was measured by lactate dehydrogenase (LDH) release with a Cytotoxicity Detection KitPLUS from Roche according to manufacturer's instructions. Control wells included wells for measuring spontaneous target cell lysis (without NK cells), and complete cell lysis (target cells lysed with 10 ul of cell lysis solution provided in the LDH kit). NK cells were purified from fresh whole human blood with human NK cell isolation kit (cat#130–092‐657) from Miltenyi Biotec according to protocol provided in the kit.
ADCC % cell killing was calculated as: $\left(\left[\text{Sample well}\right]\mathrm{Abs}49\text{0nm}–\left(\text{Sample well with}\mathrm{no}\text{antibody}\right)\mathrm{Abs}490\mathrm{nm}\right)]\times 100/\left(\left[\text{Complete cell lysis}\right]\mathrm{Abs}49\text{0nm}–\left(\text{Spontaneous target cell lysis with}\mathrm{no}\mathrm{NK}\text{cells}\right)\mathrm{Abs}490\mathrm{nm}]\right)$