CellGenix® GMP SCGM Stem Cell Growth Medium

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	with 20% heat-inactivated FBS (Gibco, Life Technologies, Carlsbad, CA, USA) and 1000 U/mL of Proleukin (Novartis, Basel, Switzerland) or IL-2 (Miltenyi, Bergisch Gladbach, Germany)

Protocol tips
with 20% heat-inactivated FBS (Gibco, Life Technologies, Carlsbad, CA, USA) and 1000 U/mL of Proleukin (Novartis, Basel, Switzerland) or IL-2 (Miltenyi, Bergisch Gladbach, Germany)

Publication protocol

The human NK cell line NK-92 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA, catalog number: CRL-2407™). Cells were initially thawed in a stem cell growth medium (CellGro; CellGenix, Freiburg, Germany) with 20% heat-inactivated FBS (Gibco, Life Technologies, Carlsbad, CA, USA) and 1000 U/mL of Proleukin (Novartis, Basel, Switzerland) or IL-2 (Miltenyi, Bergisch Gladbach, Germany) when available. To evaluate NK cell activity, the human erythroblast cell line from a chronic myelogenous leukemia patient K562 (LGC Promochem/ATCC, Manassas, VA, USA) was used as a target in degranulation assays and 51Cr-release assay. K562 cells were cultured with RPMI, GlutaMAX 1640 (Gibco) supplemented with 10% FBS. Cells were incubated at 37 °C and 5% CO2 with a humidity of 95% and cell numbers were determined every second day by Trypan Blue staining. NK-92 cells cultured in standard conditions were kept in between 0.3–1.0 × 106 cells/mL density, while NK-92SF cells were cultured at a concentration of 0.5–1.5 × 106 cells/mL and supplemented with 1000 U/mL IL-2 5 days per week in order to obtain optimal cell proliferation. All cell culture has been conducted in a BSL2 environment under strict antibiotic-free conditions. HEK293FT cell line (Thermo Fisher Scientific, Waltham, MA, USA) is an altered version of adherent human embryonic kidney cells, expressing SV40 large T antigen that provides high titer lentivirus production due to high expression of viral RNA. Both virus production and titration experiments were optimized with this cell line. HEK293FT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) Glutamax with 10% FBS (Gibco), 1 mM l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate solution (Sigma-Aldrich), and 0.1 mM MEM non-essential amino acids (Sigma-Aldrich).

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