Easysep negative enrichment kit

Human NK cells were enriched from Buffy-coats (EasySep negative enrichment kit, Stemcell Technologies, 19055). A total of 2 × 10⁶ cells/ml were cytospun onto slides, air-dried, fixed in Bouins for 10 min, permealized with Triton for 5 min and blocked with 1% BSA in PBS for 30 min at 4 °C. Slides were incubated with goat anti-human MIP-1α (R&D Systems, AF-270-NA; 1 : 50) and mouse anti-human GrzM (1 : 500) for 45 min. After two washes with PBS slides were incubated with Alexa 488-conjugated anti-mouse IgG1 and Alexa 649 donkey anti-goat IgG for further 45 min (both diluted 1 : 1000; Molecular Probes, Grand Island, NY, USA). Control slides were incubated with only secondary antibodies or the mismatching antibodies. After staining, specimens were washed in PBS and a drop of Dapi anti-fade (Invitrogen) was used for nucleic staining, as well as to reduce photo-bleaching. Slides were examined by confocal imaging on a Nikon CS 2, original magnification × 40 (WD 0.24 mm oil) using the NIS software (Nikon Instruments Inc., Melville, NY, USA).

Single-cell suspensions of spleen from naive WT, GrzM^−/−, pfp^−/− or GrzB^−/−mice were enriched for NK cells (NK1.1⁺/CD3⁻) by negative selection with a mouse NK cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted to purity (BDAriaII, San Jose, CA, USA). Human NK cells were enriched from Buffy-coats (EasySep negative enrichment kit, Stemcell Technologies, Vancouver, BC, Canada; 19055). Mouse or human NK cells were stimulated with 1 ng IL-12/ml and 10 ng IL-15/ml, 1 ng IL-12/ml and 5 ng IL-18/ml, or 1000 U/ml IL-2 and 1 ng IL-12/ml for 3, 6 and 24 h or for 4 and 24 h. Cells were used for surface marker staining, intracellular staining or to extract RNA for RT-PCR; supernatants were harvested for CBA assays or were analysed by ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.