Pe conjugated anti nkg2d

Cells were suspended in 1X PBS supplemented with 0.1% FBS. Cells were washed once in buffer and stained for 30 min with PE-conjugated anti-NKG2D, FITC-conjugated anti-CD3e, APC-conjugated anti-NKp46, and anti-CD19 (Biolegend, San Diego, CA, USA). After harvesting the cells, the expression of proteins was analyzed using NovoCyte flow cytometry (Agilent Technologies, Santa Clara, CA, USA) on over 10,000 cells.

Single-cell suspensions were prepared as previously described (Dou et al., 2018 (link)). For detection of NK cells and their NKG2D expression, cells were stained with FITC-conjugated anti-NK1.1 (BD Bioscience), APC-conjugated anti-CD3 (BioLegend), PE-conjugated anti-NKG2D (BioLegend) and corresponding isotype controls. For detection of NK cell-produced IFN-γ, firstly, 1.0×10⁶ cells were activated in vitro with 2 μl Leukocyte Activation Cocktail (BD Bioscience) for 5 h at 37°C, followed by incubation with FITC-conjugated anti-NK1.1, APC-conjugated anti-CD3 for 30 min. Then, cells were permeabilized and fixed using Cytofix/Cytoperm Soln Kit (BD Bioscience) for 30 min, followed by incubation with PE-conjugated anti-IFN-γ (BD Bioscience). FACS was performed on BD FACS Calibur flow cytometer (BD Biosciences, NJ, United States) or CytoFLEX (BECKMAN COULTER, United States). Data were analyzed using FlowJo (version 10, United States). For analysis of NK cell infiltration, the lymphocyte population was gated from the general diagram of FSC-SSC. The negative control was then determined from cells stained with isotype controls. The location of CD3⁺ cells was determined by CD3-stained cells, and that of NK1.1⁺ cells was distinguished by NK1.1-stained cells. The percentage of NK1.1⁺CD3⁻ cells in the population was recorded.