Accuri c6 system

Cells were fixed in 70 % ethanol for 1 h at 4°C. Repeated washes in cold PBS and RNase A treatment (100 μg/ml, Invitrogen) for 1 h at 37°C were followed by propidium iodide staining (50 μg/ml, Sigma-Aldrich). Analysis in the FL3 channel in DDM mode was performed using the BD Accuri C6 system in combination with BD CSampler software (Becton-Dickinson).

After transfection with the corresponding STAT1 vectors or siRNAs for 48 h, the cells were harvested and washed twice with cold PBS, then fixed in 75 % alcohol for 2 h at 4 °C. After washing in cold PBS three times, cells were resuspended in 1 mL of PBS solution with 40 μg of propidium iodide (Sigma) and 100 μg of RNase A (Sigma) for 30 min at 37 °C and analyzed with the BD Accuri C6 system (Becton–Dickinson, USA). The distribution of cells in different phases of the cell cycle was calculated using the Modifit LT software.

To evaluate the significance of each of nonsynonymous SNP of FUT1, transient expression experiments followed by flow cytometry analysis were performed as done with the FUT2 using an anti-H 1E3 monoclonal antibody^{19 ,24 (link)}. In addition to the FUT1 alleles containing each SNP concerned, the effects of the wild-type allele (FUT1*01), c.725T>G (FUT1*01N.09) inserted into pcDNA3.1(+) vectors were determined. Two μg of each construct together with 60 ng of the pGL3 Promoter was transfected into 2 × 10⁵ COS-7 cells (African green monkey kidney fibroblast-like cell) by means of TransIT-X2 (Mirus Bio LLC, Madison, WI). After 2 days, the cells were immunostained by using a mouse monoclonal antibody to H type 1–4 (1E3)^{24 (link)}, followed by incubation with FITC-labeled goat anti-mouse IgM (Bethyl Laboratories, Montgomery, TX), and H antigen expression was examined using a BD Accuri C6 system (Becton Dickinson, Franklin Lakes, NJ) as described previously¹⁹ . The experiments were repeated four times independently. The transfection efficiency in each experiment was determined by luciferase luminescence intensity and the similar transfection efficiency was confirmed by the intensity of luciferase light as described previously¹⁹ .

Cells were stained with fluorescence-conjugated antibodies for markers of Tregs and Th cells, including CD4, IFN-γ, IL-9, IL-17, IL-22, and Foxp3 (BD Pharmingen), according to the manufacturer’s protocols. For cytokine staining, the cells were stimulated using a Leukocyte Activation Cocktail with BD GolgiPlug™ for 6 h at 37 °C with 5% CO₂. For intranuclear staining, the cells were mixed with the fixation/permeabilization working solution and incubated for 30 min. Flow cytometric analysis of antibody-labeled cells was performed on a BD Accuri C6 system. The details were provided in the supplementary material. The numbers of cell subpopulations were calculated by multiplying the total cell number by the percentages of each subpopulation, as determined by flow cytometry.

MDA-MB-231 and MCF-7 cells were grown to 90% confluence, detached from the culture dish using trypsin with 1 mM EDTA, washed with FACS buffer (PBS with 2 mM EDTA, 2% BSA, and 0.05% NaN3), and stained with the β1 integrin primary antibody (10 µg/mL) for 30 min at room temperature. The sample was then incubated with a secondary antibody (FITC-anti-mouse IgG, 15 µg/mL) for 30 min at room temperature. The cells were washed three times with FACS buffer and analyzed for integrin expression using a BD Accuri C6 system (BD Biosciences).

SH-SY5Y cells (ATCC 2266) were cultured in DMEM medium with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 mM HEPES. For immunofluorescence microscopy analyses, cells were subcultured in glass coverslips pre-coated with l-lysine. HIV201B2 and U78Ab3 were labeled with Dylight 488 (ThermoFisher Scientific, Bremen, Germany) according to the manufacturer’s instructions. For flow cytometry analyses, cells were fixed with 4% paraformaldehyde and stained with Dylight 488-labeled mAb (10 µg/mL) at RT, and recorded using an Olympus 1 × 81 inverted fluorescence microscope (Olympus, Tokyo, Japan). For SH-SY5Y cell competition-binding assessment, cells were coincubated with Dylight 488-labeled HIV201B2 and DWEYS with and increasing molar ratio, and recorded using a BD Accuri C6 system (BD Biosciences, San Jose, CA, USA).