Anti ulbp1 rabbit polyclonal antibody gtx123021

Intracellular and cell surface ULBP1s were detected by a flow cytometer. Briefly, after digitonin‐permeabilization of cells, intracellular ULBP1 was immunostained by using anti‐ULBP1 rabbit polyclonal antibody (GTX123021; GeneTex) and PE‐conjugated goat anti‐rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) as primary and secondary antibody, respectively. Cells without digitonin‐permeabilization were also subjected to the immunostaining of cell surface ULBP1. Rabbit IgG isotype control (Cell Signaling Technology) was used as a negative control. The mean fluorescence intensity and the standard deviation were calculated from four independent experiments.
The binding of NKG2D to target cells was also detected by a flow cytometer. Briefly, target cells were subjected to the treatment of recombinant human NKG2D/Fc (R&D Systems) or human IgG₁/Fc chimera protein (R&D Systems), and then were immunostained by FITC‐conjugated goat anti‐human IgG, Fc fragment specific (Jackson ImmunoResearch Laboratories). The mean fluorescence intensity and the standard deviation were calculated from three independent experiments.

Before the co‐culture with NK‐92 cells, the culture medium of target cells was replaced with fresh medium. Subsequently, an equal volume of fresh medium containing NK‐92 cells was added to the target cells. The target cells were co‐cultured with NK‐92 cells for 2, 6 or 24 h in a 1 : 1 mixed medium (two kinds of media for each cell type). At 2, 6 or 24 h after co‐culture using several different E : T ratios, the culture media were recovered from the co‐cultured cells for the measurement of NK cell‐mediated cytotoxicity as described below.
For the blocking of NKG2D ligands, target cells were also treated with anti‐ULBP1 rabbit polyclonal antibody (GTX123021; GeneTex, Irvine, CA, USA) or anti‐ULBP2/5/6 antibody (R&D Systems, Minneapolis, MN, USA) for an hour before the co‐culture with NK‐92 cells. Subsequently, NK cell‐mediated cytotoxicity was measured as described below.