Mojosort human nk cell isolation kit

Primary human NK cells were isolated from peripheral blood of healthy donors, for which they gave written informed consent according to the protocol (#2019-623) approved by the local ethics committee (Heinrich Heine University, Düsseldorf, Germany). Ficoll density gradient centrifugation (Ficoll-Paque Plus; Cytiva Europe, Freiburg, Germany) was performed to collect peripheral blood mononuclear cells (PBMCs). NK cells were then enriched by immunomagnetic negative selection using MojoSort™ Human NK Cell Isolation Kit (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. NK cell purity and phenotype were determined by flow cytometry (MACSQuant^® Analyzer 10, Miltenyi Biotec, Bergisch Gladbach, Germany) on days 0, 7, and 14, using fluorochrome-conjugated antibodies against CD3, CD33, CD56, and CD94 (all REAfinity™ clones from Miltenyi Biotec). NK cells were activated and expanded in NK MACS^® Medium (Miltenyi Biotec) supplemented with 5% heat-inactivated human AB serum (Sigma-Aldrich, Darmstadt, Germany), 1% P/S, 500 IU/ml of IL2 (Proleukin, Novartis, Basel, Switzerland), and 10 ng/ml of IL15 (Miltenyi Biotec) at a concentration of 1–2 million cells/ml.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy, anonymous donors provided by the Luxembourg Red Cross. Upon receipt, buffy coats were diluted ten times with Ca²⁺/Mg²⁺ free phosphate buffered saline (PBS) and the low-density PBMC fraction was isolated by centrifugation over a Lymphoprep density gradient (Stemcell Technologies, cat. # 07861). After centrifugation, the PBMC layer was collected, washed several times with Ca²⁺/Mg²⁺ free PBS and red blood cells were lysed with ACK buffer (ThermoFisher Scientific, cat. # A1049201). Following erythrocytes lysis, cells were washed once with Ca²⁺/Mg²⁺ free PBS, counted with Trypan blue and cell concentration adjusted for NK cell isolation. NK cells were isolated with the MojoSort human NK cell isolation kit (BioLegend, cat. # 480054) combined with a LS column (Miltenyi Biotec, cat. # 130-042-401). Isolated NK cells were cultured overnight in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES (ThermoFisher Scientific, cat. # 15630056), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 100 U/mL recombinant human interleukin-2 (IL-2; Peprotech, cat. # 200-02) and 10 ng/mL recombinant human IL-15 (IL-2; Peprotech, cat. # 200-15).

Isolation of pure NK cells was performed with MojoSort™ Human NK Cell Isolation Kit (Biolegend). PBMC or enriched NK cells were cultured in NK MACS medium [1% NK MACS supplement, 5% human AB serum (Sigma Aldrich), 500 U/ml IL-2 (Proleukin), and 25ng/ml IL-15 (Miltenyi Biotec)]. NK cell expansion with stimulator cells was performed as previously described (10 (link)). Briefly, PBMCs (1.5×10⁶) were incubated in 24-well plates with 1×10⁶ irradiated (40 Gy) K562-mb15-41BBL cells or K562-mb15-mb21-41BBL in SCGM CellGro Medium (CellGenix, Freiburg, Germany) supplemented with 10% FBS and 1% P/S and 40 U/ml human IL-2 (Proleukin).