Af 270 na

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).

Recombinant full-length human GST-tagged MIP-1α, into which a processing consensus site for human GrzM (Valine Leucine Phenylalanine) was encoded in the polylinker region (Abnova, P01), was incubated with human recombinant GrzM or a S/A mutant (inactive) GrzM for the indicated times at 37 °C, or left on ice as a control. Products were separated on SDS-polyacrylamide gel and immunoblotting was performed as described previously using goat anti-human MIP-1α (R&D Systems, AF-270-NA) Blots were developed with ECL using the AP substrate CDP-Star (Applied Biosystems, Carlsbad, CA, USA).