Nk1.1 inkt cell isolation kit

A single-cell suspension from the spleen was harvested and resuspended in RPMI complete medium consisting of RPMI 1640 (Gibco BRL, USA) medium supplemented with 10 mM HEPES, 2 mM L-glutamine, 10% FBS, 5 mM 2-mercaptoethanol, and 100 units/mL penicillin-streptomycin. iNKT cells were enriched using NK1.1⁺ iNKT cell isolation kit (Miltenyi, Germany). The enriched iNKT cell population was >88% pure after MACS. CD11c⁺ DCs and CD4⁺ T cells were isolated from mice using a MACS system (Miltenyi, Germany). CD11c⁺ DC population was >94% pure after MACS and the CD4⁺ T cell population was >94% pure.

Murine NKT cells were isolated from the spleen of 10 weeks old Mif^−/− mice according to the protocol of a NK1.1 iNKT cell isolation kit (Miltenyi Biotec; Bergisch Gladbach; Germany) with magnetic beads mediated cell separation. After isolation, cells were resuspended in RPMI supplemented with 10% FCS, penicillin-streptomycin (100 U/mL), 15 mM Hepes, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol (Invitrogen, Carlsbad, CA, USA) and plated on plastic dishes.
Cells were stimulated with 50 ng/mL recombinant murine MIF prepared as described previously [35 (link)] or recombinant murine IL-12 (PeproTech, Rocky Hill, NJ, USA) for 24 h.

A single-cell suspension of splenocytes was prepared and resuspended in RPMI complete medium, consisting of RPMI 1640 (Gibco BRL, Gaithersburg, MD, USA) medium supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 units/mL penicillin–streptomycin, and 5 mM 2-mercaptoethanol. iNKT cells were enriched using an NK1.1⁺ iNKT cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions [18 (link)]. The NKT cell population was >89% pure among all MACS-purified populations.

A single-cell suspension of splenocytes was prepared and resuspended in RPMI complete medium consisting of RPMI 1640 (Gibco BRL, USA) medium supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 units/mL penicillin-streptomycin, and 5 mM 2-mercaptoethanol. NK and iNKT cells were enriched using the NK cell isolation kit II and NK1.1 iNKT cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions, respectively. The NK population was >87% pure and NKT population was >91% pure after MACS. In addition, for the preparation of CD11c⁺ total DCs, whole splenocytes from WT B6 mice were stained with anti-CD11c monoclonal antibody (mAb) for MACS and enriched for CD11c⁺ DCs by positive selection. The DC population was >95% after MACS. Bone marrow-derived basophils (BM basophils) were separated as follows: IL3-cultured BM cells were stained with biotin-conjugated anti-CD49b (clone DX5) mAbs, and then DX5⁺ cells were positively selected using anti-biotin MACS beads. The basophil population was >92% after MACS.