Sytox blue stain

1 × 10⁵ cells were incubated for 30 min with anti-Integrinβ1 (Santacruz Biotech, Dallas, TX, USA) or anti-Integrinα2 (BD Horizon, Franklin Lakes, NJ, USA) antibodies, and with the secondary antibody anti-mouse Alexa-fluor 488, for another 30 min. For PDL1 (Biolegend, San Diego, CA, USA), after 24 h IFN-γ treatment, 1 × 10⁵ cells were incubated for 30 min in the same buffer with anti-PDL1 antibody. Dead cells were excluded by Sytox Blue Stain (Life Technologies, Carlsbad, CA, USA) or propidium iodide (Sigma, St. Louis, MO, USA). For cell cycle analysis, the cells were fixed with 70% ethanol, washed three times with PBS and stained for 3 h at room temperature with PBS–propidium iodide, then analysed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and FCS express 5 (De Novo software, Glendale, CA, USA).

For the detection of NKG2DLs, single-cell suspensions of monolayer cells or tumor spheroids treated with TrypLE™ Express (Life Technologies, USA) were stained with mouse monoclonal antibodies: mouse anti-human MICA (AMO1[36 (link)]), mouse anti-human MICB (MAB1599), mouse anti-human ULBP1 (MAB1380), mouse anti-human ULBP2 (MAB1298) and mouse anti-human ULBP3 (MAB1517, all R&D Systems, USA). Rat anti-mouse IgG₁-APC (130-095-902, Miltenyi Biotec, Germany) or rat anti-mouse IgG_2a/b-APC (130-095-880, Miltenyi Biotec, Germany) served as secondary antibodies. As negative control, samples incubated with secondary antibodies only were used. Cells were analyzed with FlowJo software (Tree Star, USA) after measurement on a FACS Canto II instrument equipped with a 96-well plate HTS Sampler. Viability of cells was analyzed by SytoxBlue stain (Life Technologies, USA). 10,000 events of viable cells were analyzed. To calculate x-fold MFI over background, data of individual experiments (n = 3) were normalized by division of the MFI by the MFI of the secondary antibody controls to calculate mean ± SEM.

Cells were detached and incubated with APC-conjugated CD44 antibody (BD-PharMingen, San Diego, CA, USA 559942) or isotype control APC-conjugated IgG2B (BD-PharMingen) in PBS BSA 1% (Sigma) for 30 minutes on ice prior to flow cytometric analysis. Sytox Blue Stain (Life Technologies, Eugene, OR, USA S34857) was added to exclude dead cells. For CD44v8-10 staining 1×10⁵ cells were incubated with 3 μg/ml anti-human CD44v9 primary antibody (clone: RV3) (Cosmo Bio Co. Ltd, Tokyo, Japan LKG-M001) in PBS 0.2 % BSA for 45 minutes at 4°C. Cells were then incubated with the secondary antibody APC-labeled Goat anti-Rat IgG (H+L) (Invitrogen, Carlsbad, CA, USA A10540) for 30 minutes at 4°C. After washing, cells were assayed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data analyzed employing FCS5 express Software (De Novo Software). For cell cycle analysis, after labeling for CD44v9, the cells were fixed with 70% ethanol, a few drops at a time mixing the cells, and incubated on ice for 30 minutes. The cells were centrifuged at 500 x g for 10 minutes, washed once in PBS by centrifugation and resuspended in 1 ml PBS containing 5 μg/ml of propidium iodide. Samples were assayed by CyAn ADP flow cytometer and analyzed employing FCS5 express Software.