Mab1298

The following primary antibodies were used for flow cytometry: anti-MICA (R&D, MAB1300), anti-MICB (R&D, MAB1599), anti-ULBP1 (R&D, MAB1380), anti-ULBP2 (R&D, MAB1298, with cross reactivity to ULBP5 and ULBP6), anti-ULBP3 (R&D, MAB1517), anti-GLUT-1 (abcam, Ab150299), anti-IFNƔ (R&D, MAB285), anti-CD56 (BioLegend, 36503), anti-CD3 (ThermoFisher Scientific, 56-0038-82), anti-σ1 (Sigma-Aldrich, MAB994-I-25UG), anti-CD4 (BioLegend, 344604), anti-CRT (Invitrogen, 902A), anti-ERp57 (BioLegend, 937301) and anti-PVR (BioLegend, 337602). Additionally, anti-KIRL2D3, murine anti-NKG2D, and anti-NKP46 antibodies were purified from hybridomas as previously described (48 (link)). These primary antibodies were stained with AF448-coupled donkey anti-mouse IgG (Jackson ImmunoResearch, 715-545-151) or APC-coupled goat anti-mouse IgG (Thermo Fisher Scientific, 31981). Murine NKG2D-Ig and human NKG2D-Ig fusion protein (R&D, 1299-NK) were followed by secondary staining with PE-coupled F(ab’)₂ fragment goat anti-human IgG (Jackson ImmunoResearch 109-116-088). Additionally, a Fixable Viability Dye eFluor™ 780 (FVD) (Thermo Fisher Scientific, 65-0865-14) was included in the flow cytometry staining to exclude the dead population.

For the analysis of NKG2D ligand expression, cells were washed twice and incubated with 5mM EDTA in PBS for 5 min to detach the cells. Spheroids were also treated with 5mM EDTA and manually disrupted to achieve a single cell suspension. Cells were then resuspended in PBS supplemented with 0.03% azide with 5% BSA for blocking. Staining was performed in the same buffer using monoclonal antibodies against MICA (2C10, Santa Cruz, Santa Cruz, CA, USA), MICB (mAb 1599, R&D System, Minneapolis), ULBP1 (mAB 170818, R&D System, Minneapolis), ULBP2 (MAB1298, R&D System, Minneapolis), ULBP3 (Clone 166510, R&D System, Minneapolis), HLA-A, B, C (W6/32, Thermo Fisher, UK), or IgG1, isotype control monoclonal antibodies (eBiosciences, San Diego, CA, USA). Cells were then washed twice and labeled with FITC-labeled polyclonal rabbit anti-mouse IgG (STAR9B, Serotec, Raleigh, NC, USA) or goat anti-mouse IgG Cy5 conjugated (AP124S, Chemicon). Propidium iodide (Sigma) was added at a concentration of 50 µg/ml to identify viable cells. Flow cytometry was performed using a BD^®FACSCanto machine and FACSDiva Software (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star).