Novoexpress software 1

The RAW264.7, J774A.1 and JAWS II cells (1 × 10⁵ cells/well in 24-well plates) were incubated with boron carbide preparations at concentrations 10, 50, 100 and 200 µg/ml, and BMDM (1.2 × 10⁵ cells/well) at concentrations 10, 50 and 100 µg/ml for 24 h. After this time, 32 µM bromodeoxyuridine (BrdU; Becton Dickinson) solution was added for 1 h, then collected and centrifuged for 10 min at 500 × g. The pellets were suspended in 70% ethanol and stored at -20 °C.
For BrdU staining, fixed cells were centrifuged (5 min, 500 × g, 4 °C) and incubated with 2 M HCl, 0.5% Triton X-100 for 30 min at room temperature. Next, the suspension was centrifuged (10 min, 500 × g, 4 °C), resuspended in 0.1 M Na Borate pH 8.5 and centrifuged again (10 min, 500 × g, 4 °C). Pellets were resuspended in PBS with 1% FBS (Biowest), 0.5% Tween20 (Sigma-Aldrich) and 20 µg/ml ribonuclease A (RNase; Sigma-Aldrich) and stained with anti-BrdU-FITC antibody (Becton Dickinson) for 30 min at room temperature. After this time, cells were centrifuged (10 min, 500 × g, 4 °C) and resuspended in PBS with 5 µg/ml propidium iodide (Invitrogen). Samples were analyzed using flow cytometer LSRFortessa with Diva Software (Becton Dickinson). The scheme of flow cytometry analysis was performed using NovoExpress software 1.3.0 (ACEA Biosciences, Inc.). Cell cycle assays were conducted in three repetitions.

Boron carbide preparations at a concentration of 100 µg/ml were incubated with the cultured RAW264.7, J774A.1, JAWS II cells and BMDM (0.1 × 10⁶ cells/well) in 24-well plates for 24 h. After this time, the changes in size and granularity of cells were analyzed in triplicate for each sample based on forward scatter (FSC) versus side scatter (SSC) using flow cytometer LSRFortessa with Diva Software (Becton Dickinson). Flow cytometric dot plots were prepared using the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

The expression of EGFP in genetically modified DC was measured 24 h after cell transduction by the BD FACSDiva software version 8.0 and the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

Splenocytes, lymph node cells, and tumor cells isolated from mice were incubated with anti-mouse CD16/CD32 mAb (15 min, 4°C, eBioscience). Splenocytes were stained with the LIVE/DEAD FixableViolet Dead Staining Kit (Thermo Fisher Scientific, Inc.). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, CD19 PE-CF594, and CD49b PE-CF594 (all from BD Biosciences); CD45 V500, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 Alexa Fluor 700, CD86 PE-Cy7, and MHC II APC-Cy7 (all from BioLegend) for myeloid cell identification; and CD45 V500, CD4 PerCp-Cy5.5, CD8 PE-Cy7, CD49b PE-CF594, CD44 PE, and CD62L BV605 for lymphocyte identification. Cells were incubated with antibodies for 45 min at 4°C. Additionally, the percentage of Treg cells was determined among the splenocytes. For this purpose, the cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and then incubated with anti-FoxP3 APC antibodies (eBioscience) [28 (link)]. To identify dead cells in lymph node cells and tumor cells, 50 μl of DAPI dye solution (1 μg/ml, Molecular Probes) was added to the samples immediately before analysis. The analysis was performed using a BD LSRFortessa Cell Analyzer (Becton Dickinson, Cat. No. 649225B5) with the BD FACSDiva software 8.0 and the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

Target MC38 cells were labeled with DiOC18(3) (20 min, 37°C, Invitrogen) and applied to round-bottom 96-well plates (Greiner) at a density of 1 × 10⁴ cells/well. Five-day stimulated splenocytes were plated on target cells at a density of 1 × 10⁵ cells/well (ratio of effector cells to target cells was 10 : 1) or a density of 3 × 10⁵ cells/well (ratio of effector cells to target cells was 30 : 1). The rh IL-2 was added to each well (200 U/ml, ImmunoTools). After 4 hours, dead cells were stained with 7.5 nM propidium iodide solution (10 min, 37°C, Sigma-Aldrich). Samples were analyzed by a BD LSRFortessa Cell Analyzer (Becton Dickinson, Cat. No. 649225B5) with the BD FACSDiva software 8.0 and the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.). The effector cell cytotoxic activity was determined as the percentage of dead target cells minus the percentage of dead control cells.

The RAW264.7 and J774A.1 cells (0.5 × 10⁵ cells/well for 24 h incubation and 0.25 × 10⁵ cells/well for 72 h in 24-well plates), JAWS II cells (1 × 10⁵ cells/well for 24 h incubation and 0.5 × 10⁵ cells/well for 72 h in 24-well plates) were incubated with boron carbide preparations at concentrations 10, 50, 100 and 200 µg/ml, and BMDM (1.2 × 10⁵ cells/well for both times) at concentrations 10, 50 and 100 µg/ml for 24 and 72 h. After this time, cells were harvested, suspended in a binding buffer and centrifuged (10 min, 100 × g, 4 °C). Next, cells were stained with Annexin V protein conjugated with APC fluorochrome (Becton Dickinson) for 15 min at room temperature. BMDM were additionally stained with anti-F4/80 BV421 (BioLegend) for 45 min at 4 °C. To assess the percentage of dead cells, 10 µg/ml propidium iodide (PI; Invitrogen) was added. The cells were analyzed using flow cytometer LSRFortessa with Diva Software (Becton Dickinson). Scheme of flow cytometry analysis was performed using NovoExpress software 1.3.0 (ACEA Biosciences, Inc.). Apoptosis assays were performed in two independent experiments, each in triplicate.

The flow cytometry method was applied to determine the percentage and mean fluorescence intensity of CD11c, MHC II, CD80, and CD86 on the surface of living DCs. Cells were labeled with monoclonal antibodies conjugated with fluorochromes: anti-CD11c BV650, anti-MHC II APC-Cy7, anti-CD80 PerCP-Cy5.5, and anti-CD86 PE-Cy7 all from BioLegend. The cells were stained for 45 min at 4°C. To identify dead cells, 50 μl of DAPI dye solution (1 μg/ml, Molecular Probes) was added to the samples immediately before analysis. The expression of cell surface markers was analyzed using a BD LSRFortessa Cell Analyzer (Becton Dickinson, Cat. No. 649225B5) with the BD FACSDiva software version 8.0 and the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).