Anti gr1

TRI reagent was from Sigma-Aldrich (Madrid, Spain). Antibodies for the detection of murine CD135 (Flt3), CD34, CD16/32, and CD127 (IL-7Rα) were from eBioscience (Barcelona, Spain). The Lineage Cell Depletion Kit for mice, FcR Blocking reagent and murine flow cytometry antibodies (CD3e, CD4, CD8a, CD11b, CD19, CD43, CD45R (B220), CD117 (c-Kit), anti-Gr1, anti-Sca-1, anti-Ter-119, anti-IgM) were from Miltenyi Biotec (Madrid, Spain). Super Script II reverse transcriptase and RNase OUT ribonuclease inhibitor were from Invitrogen and Thermo Fisher Scientific (Madrid, Spain). GoTaq^R qPCR master mix was from Promega (Madrid, Spain).

Single cell suspensions of splenocytes or peripheral blood were prepared using the standard techniques. Fc receptors were blocked by using 2.4G2 mAb prior to surface staining with the indicated antibodies. The negative selection of NK cells was determined by staining with cell surface monoclonal antibodies (mAbs), including anti-CD19, anti-CD4, anti-CD8a, anti-CD5, anti-Gr1, and anti-Ter-119 (Miltenyi Biotech). The surface phenotype of NK cells was determined by staining with mAbs, including anti-CD3-PE and anti-NK1.1-APC (Biolegend, San Diego, CA). Intracellular IFNγ was measured by pacific blue-conjugated anti-IFNγ (Biolegend) as described previously [47 (link)]. Dead cells were excluded by propidium iodine (PI) staining and live cells were gated on PI-negative cells. The NK cells of donors or recipients were distinguished by using anti-CD45.1 mAb (A20, BD Biosciences, San Jose, CA), FITC-34-2-12 (H-2D^d mAb, BD Biosciences), or Bio-KH95 (anti-H-2D^b mAb, BD Biosciences). Data acquisition was performed with FACS Calibur flow cytometer (BD Pharmingen, San Jose, CA) using a CellQuest software (BD Biosciences). Data analysis was performed using a FlowJo software [48 (link)].