Summit version 4

MC38 or CT-26 cells were cultured with 100 ng/ml rmIL31 or vehicle control for 48 hours. Cells were detached and re-suspended in 100 μl PBS. Cells were fixed by slowly adding the suspension to a tube containing 1 ml 70% ethanol with gentle vortexing. Cells were incubated on ice for 20 min, centrifuged and re-suspended in 1ml of Propidium Iodide (PI) master mix containing 40 μg/ml PI (BioLegend, Inc., USA), 10 μg/ml RNase (Sigma-Aldrich), and 950 μl PBS. After a 30 min incubation at room temperature, samples were analyzed with a Cyan-ADP flow cytometer and Summit Version 4.3 software (Beckman Coulter, Switzerland) using linear scale histogram.

MC38 or CT-26 cells were cultured with 100 ng/ml rmIL31 or vehicle control for 48 hours. The cells were detached and re-suspended in 60 μl Annexin V Binding Buffer (MBL, Japan). Subsequently, 2.5 μl of Annexin V-FITC antibody (Bio Legend, Inc., USA) and 2.5 μl of 7-Aminoactinomycin D (7-AAD) (Sigma-Aldrich, USA) were added and samples were incubated for 15 min at RT in the dark. Annexin V Binding Buffer (300 μl) was then added to each sample. Samples were analyzed with a Cyan-ADP flow cytometer and Summit Version 4.3 software (Beckman Coulter, Switzerland).

For HER-2 cell surface staining with trastuzumab antibody (Herceptin; Roche, Basel, Switzerland), titration assays were performed by indirect immunofluorescence using as targets SKBR3, BT474 and MCF-7 cell lines. In particular, 2 × 10⁵ cells/sample were incubated with different concentrations (2, 0.2, 0.02, 2 × 10⁻³ and 2 × 10⁻⁴ µg/ml) of the mAb for 30 min at 4 °C. After washing in FACS buffer, an Alexafluor 647-conjugated goat anti-human IgG secondary antibody (Molecular Probes, Inc. Eugene, OR, USA) was added and incubated for 30 min at 4 °C. After 2 washes, the cells were analyzed by flow cytometry on a CyAn-ADP-Flow-Cytometer (Beckman-Coulter); 5000 cellular events were analyzed per sample. Data were analyzed using Summit version 4.3 Software (Beckman-Coulter).
Results were expressed as mean ratio of relative fluorescence intensity (MRFI), calculated as follows: mean fluorescence intensity (MFI) of HER-2 staining/MFI of secondary antibody staining.

Cell suspensions were assessed for T cells, macrophages, neutrophils and their subpopulations thereof using flow cytometry, as described previously [24 (link)]. Briefly, tumors removed from tumor-bearing mice were prepared as single-cell suspensions as previously described [35 (link)], and then immunostained with the following antibody mixture: CD3ε for T cells, F4/80 for macrophages, CD11b, Ly6G and Ly6C for neutrophils. Mature neutrophils were identified as CD11b⁺Ly6C^(high)Ly6G⁺, and immature neutrophils as CD11b⁺Ly6C^(low)Ly6G⁺. Flow cytometry experiments were performed using BD LSRFortessa™ (BD Bioscience) flow cytometer and analyzed with Summit version 4.3 (Beckman Coulter).