Facscanto 10

Spleen neutrophils from mice were incubated for 4 h at 37° C in RPMI medium supplemented with 10% FBS and Golgi Stop (BD Biosciences). Nonspecific sites were saturated in the FCSB solution (eBiosciences) and then incubated with the membrane antibodies (CD11b, CD45, Ly-6G) in the FCSB solution. After fixation and permeabilization (Fixation/Permeabilization solution, BD Biosciences), neutrophils are incubated with an anti-Granzyme-B. The cells were analyzed by flow cytometry on BD FACSCanto™ 10.

The flow cytometric measurements were performed using FACSCanto 10 equipped with FACS Diva software for data acquisition and analysis (BD Biosciences). Cytometer setup and tracking beads (CST, BD Biosciences) were used for daily quality control and setup of initial PMT voltages. On the first day of experiment, PMT voltage of the PE channel was optimized to ensure the MedFI of unstained cells is approximately 2.5 times above the electron noise of the PE channel according to previously published protocol [45 (link)]. QuantiBrite PE beads were then run; the resulting MedFI values of the four PE intensity beads were recorded and used as target values to ensure consistency between experiments performed at different days. If needed, the PMT voltage of PE channel was slightly adjusted to ensure that the target MedFI values were achieved for instrument performance harmonization.

MICA antibodies or IC were conjugated with CypHer5E Mono NHS Ester (Ge Healthcare, cat. no. PA15401), as recommended by the supplier. MICAB1-Cypher5 or IC-Cypher5 at 1 µg/mL was added to 50 000 Raji wt or Raji MICA*001 cells, and the mixture was incubated for 30 minutes, 1 h, 4 h or overnight at 37°C. Internalization was stopped by placing the plate on ice for 10 minutes. The cells were washed and resuspended in cold PBS containing the mortality marker Sytox blue (Thermo Fisher Scientific, cat. no. S34857) diluted 1/10000. Cells were acquired and analyzed on a FACS CANTO10 (BECTON DICKINSON (BD)) flow cytometer, with FACS Diva software.

For the DR-GFP homologous recombination assay (32 (link)), GFP-positive cells were detected by flow cytometry 3 days after I-SceI transfection. For the DR-GFP experiments using TGFβ pathway modulators, the TGFβ receptor inhibitor LY2109761 (8 μM) or the TGFβ1 peptide (5 ng/ml) were added 24 h before I-SceI transfection, as well as for the 3 days after transfection. For the neutral comet assay, cells were pre-treated with TGFβ1 (5 ng/ml) for 24 h. Cells were then incubated in fresh media containing the indicated concentrations of olaparib or cisplatin, as well as TGFβ1 (5ng/ml), for 24h. Cells were then processed using the Comet Assay Kit (Trevigen, 4250-050) according to the manufacturer's instructions. Olive tail moment was analyzed using CometScore 2.0. BrdU alkaline comet assay was performed as previously described (34 (link)). For cell-cycle analyses, cells were fixed in 70% ethanol and stained with propidium iodide. Cell-cycle profiles were read on a BD FACSCanto 10 instrument and analyzed using FlowJo software.