Mouse flt3l

NK cell differentiation from HPCs was performed as previously described. In brief, to isolate HPCs, Lin⁻ (B cell (B220), T/NK cells (CD2), granulocytes (Gr-1), monocytes (CD11b), NK/NKT cells (NK1.1), and erythrocytes (TER-119)) cells were purified using the MACS Cell Seperation kit (Miltenyi Biotec). c-Kit⁺ cells from the Lin⁻ cells were positively selected using the CD117 (c-Kit) microbeads (Miltenyi Biotec). The purified HPCs were plated into a 24-well plate (BD) at 1 × 10⁶ cells/well and cultured for 7 d in complete RPMI1640 medium supplemented with a mixture of mouse Flt3L (50 ng/ml, Peprotech), mouse SCF (30 ng/ml, Peprotech), mouse IL-7 (0.5 ng/ml, Peprotech), Indometacin (2 ug/ml, Sigma), and gentamycin (20 ug/ml, Sigma). The cell were refreshed with the same media on day 3. To generate the mNK cells, 7 d-HPCs were maintained with IL-15 (30 ng/ml, Peprotech) for 6~7 days.

Growth time was 4 days. The medium was IMDM (Thermo Fisher Scientific), 20% FCS (Thermo Fisher Scientific), 1% penicillin-streptomycin-glutamine (Gibco), 10 ng ml⁻¹ mouse GM-CSF (PeproTech), 10 ng ml⁻¹ mouse SCF (PeproTech), 5 ng ml⁻¹ mouse G-CSF (PeproTech), 5 ng ml⁻¹ mouse interleukin-3 (IL-3) (PeproTech), 5 ng ml⁻¹ mouse interleukin-6 (IL-6) (PeproTech), 5 ng ml⁻¹ mouse interleukin-5 (IL-5) (PeproTech), 5 ng ml⁻¹ mouse Flt3L (PeproTech), 2 ng ml⁻¹ mouse TPO (PeproTech) and 2 U ml⁻¹ Epo (R&D Systems).

Growth time was 2 days. The medium was IMDM, 20% FCS, 1% penicillin-streptomycin-glutamine (Gibco), 10 ng ml⁻¹ mouse GM-CSF (PeproTech), 10 ng ml⁻¹ mouse SCF (PeproTech), 5 ng ml⁻¹ mouse G-CSF (PeproTech), 5 ng ml⁻¹ mouse IL-3 (PeproTech), 5 ng ml⁻¹ mouse IL-6 (PeproTech), 5 ng ml⁻¹ mouse IL-5 (PeproTech), 5 ng ml⁻¹ mouse Flt3L (PeproTech).