Facsdiva software

The uptake of DU-145 released antigens by DCs was first investigated. Briefly, DU145 cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (1 μM; Life Technologies Thermo Fisher, CA, USA) and incubated for 20 min at 37°C protecting from light. After two washes in PBS (37°C), the cells were treated with 2.5 μg/ml of Cabozantinib (48 h). Cell culture supernatants were collected and added to monocyte-derived DCs at the immature stage for 48 h. At the end of the incubation, the iDCs samples were acquired by a FACSCanto II flow cytometer (Becton Dickinson) and analyzed by FACSDiva software (Becton Dickinson) to verify the transfer of CFSE to the cytoplasm of the cells.
To study the endocytic capacity of DCs cultured in the presence or in the absence of 2.5 μg/ml Cabozantinib, FITC-dextran (1 mg/ml; Molecular Probes, Eugene, USA) was added to DCs for 1 h at 37°C and 4°C. After washing, cells were acquired by FACSCanto II flow cytometer (Becton Dickinson) and analyzed by FACSDiva software (Becton Dickinson).

Cells were detached with Accutase Cell Detachment Solution (Beckton Dickinson) and washed in PBS. Required cell number (1 × 10⁶) was resuspend in cold Stain Buffer (Beckton Dickinson) and used for further flow cytometry analysis. Cell markers were analyzed with Human MSC Analysis Kit (Beckton Dickinson) containing antibodies conjugated with fluorochrome against following antigens: CD73, CD90, CD105 (positive markers), CD11b, CD19, CD34, CD45, and PE (negative markers) (Table 1). Cells were incubated in diluted antibodies in the dark for 30 min. After incubation, cells were washed twice with Stain Buffer (Beckton Dickinson) and resuspend in Stain Buffer. Resuspended cells were analyzed using FACS Canto II (Beckton Dickinson) with FACSDiva Software (Beckton Dickinson) and FlowJo 10 (Beckton Dickinson).
In 3rd and 10th div of 3D culture, spheroids were dissociated and resuspended in Stain Buffer. Before running the sample, cells were filtered through 30 µm filter (Miltenyi Biotec, Bergisch Gladbach, Germany) to avoid dublets. Cells were analyzed to compare the change in size with flow cytometry FACS Canto II (Beckton Dickinson) with FACSDiva Software (Beckton Dickinson) and FlowJo 10 (Beckton Dickinson).

All samples were analysed with a flow cytometer (FACSCanto II cytometer with FACSDiva software, BD Biosciences, San Jose, CA, USA) to assess cell cycle and apoptosis. The experiments were performed in three replicates. Viability of the cells was estimated using the Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK) according to the recommended protocol. Briefly, harvested cells were washed with PBS, incubated with FITC-Annexin V and propidium iodide (PI) for 5 min at room temperature and analysed. Final apoptotic cell number was estimated as a total percentage of early apoptotic cells staining positive for Annexin V and negative for PI and late apoptotic cells positive for both Annexin V and PI.
For the cell cycle distribution patterns, 3 × 10⁵ CGTH cells harvested by trypsinization were washed with PBS and fixed in 70% cold ethanol at −20 °C overnight. Following fixation, cells were washed twice with PBS and stained with 5 µg/mL of propidium iodide in PBS containing 0.1% NP-40 and 10 µg/mL of DNase-free RNase. After 30 min at room temperature in the dark, the cell cycle distribution was analysed on a FACSCanto II cytometer with the FACSDiva software (BD Biosciences, San Jose, CA, USA). Relative percentages of cells in respective cell cycle stages were estimated using the ModFit LT 4.1.7 software (Verity Software House, USA).