Fcs express 4 flow software

To detect cell surface expression of IL-23R, activated spleen cells from IL-23R, IL-23R-Y416F and IL-23R-Y416FΔICD mice were washed with FACS buffer (PBS containing 1% BSA) and incubated with 5 × 10⁵ cells/100 μl FACS buffer supplemented with a 1:100 dilution of IL-23R antibody, (cat. 150903, Biolegend, San Diego, CA, USA) for 1 h on ice. Finally, cells were washed once with FACS buffer, suspended in 500 μl FACS buffer and analyzed by flow cytometry (BD FACSCanto II flow cytometer, BD Biosciences, San Jose, CA, USA). Data was evaluated using the FCS Express 4 Flow software (De Novo Software, Los Angeles, CA, USA).

For the detection of the SyCyR cell surface expression of stably transfected Ba/F3-gp130 cells, cells were washed in FACS buffer (PBS, 1% bovine serum albumin). 5x10⁵ cells were resuspended in 50 μl FACS buffer containing specific primary antibodies as indicated (myc 1:100; HA 1:1000). Cells were incubated for 1 h at room temperature and then washed with FACS buffer and resuspended in 50 μl FACS buffer with Alexa Flour 488 conjugated Fab goat anti-rabbit IgG (cat. #A11070) in a 1:500 dilution and again incubated for 1 h at room temperature. Cells were washed with FACS buffer, resuspended in 500 μl FACS buffer and analyzed by flow cytometry (BD FACSCanto II flow cytometer, BD Biosciences, San Jose, CA, USA). Data was evaluated using the FCS Express 4 Flow software (De Novo Software, Los Angeles, CA, USA).

The analysis method essentially followed the reported protocol^{43 (link)}. Tumor cells were seeded in 6-well microplates at 1.2 × 10⁵ cells per well for 12 h, followed by treatment with various biobutyrate for 72 h. Cells were harvested with trypsin (Invitrogen), washed with PBS twice, and fixed with ice-cold ethanol (70%) for 2 h at − 20 °C. Fixed cells were washed twice in cold PBS and resuspended in 300 μL of freshly prepared PBS plus 0.1% Triton X-100, 0.2 mg/mL DNase-free RNase A (Sigma-Aldrich), and 10 μg/mL propidium iodide (PI) (Roche, IN, USA). After incubation at 37 °C in the dark for 20 min, cells were filtered through nylon mesh (BD Biosciences, CA, USA). PI-stained cells were loaded and analyzed by the FACScanto flow cytometer system (BD Biosciences) with the excitation at 536 nm and emission at 617 nm. Data were analyzed by using the FCS Express 4 Flow Software (De Novo Software).

Intracellular content of rhodamine 123 (Sigma-Aldrich, St. Louis, MO, USA) was evaluated by flow cytometry, as previously reported [36 (link),37 (link)]. The working rhodamine 123 solution was freshly prepared prior to each experiment by dissolving 1 mg in distilled water and then diluting in a complete culture medium to the final concentration of 5 µM. Briefly, the treated and control cells were removed from the culture flask using TrypleTM Express solution (GIBCO, Waltham, MA, USA), spun down, and pelleted. The cells were then resuspended in 1 mL of rhodamine 123 solution in plastic Falcon tubes. The samples were incubated for 1 h at 37 °C in a CO₂-incubator. Following the incubation time, the cells were washed once with ice-cold HBSS (4 °C) (Hank’s Balanced Salt Solution, Lonza, Basel, Switzerland) and then resuspended in 0.5 mL of ice-cold HBSS. The samples were immediately analyzed with a CyFlow^® SPACE flow cytometer (Sysmex, Kobe, Prefektura Hyōgo, Japan) using 488 nm (50 mW) laser excitation and a 536/40 (BP) filter for rhodamine fluorescence detection. The results were analyzed using FCS express 4 flow software (De Novo Software, Glendale, CA, USA). Results were expressed as a mean fluorescence intensity (MIF) and normalized to the control (E0) according to previous study [37 (link)]. Experiments were repeated a minimum of three times.

In all tests performed, the samples of cells were analyzed with CyFlow® SPACE flow cytometer (Sysmex-Partec, Görlitz, Germany) with laser excitation and BP filters recommended for a specific test method, as will be detailed below in the description of procedures for each test performed. The results were analyzed with FCS express 4 flow software (De Novo Software, Glendale, CA, USA).

Apoptosis assays were performed using Alexa Fluor 488- or Pacific Blue-conjugated Annexin V/Dead Cell Apoptosis Kit (A13201 or A35122, Molecular Probes, Crand Island, NY), as instructed by the manufacturer, and quantified using an LSR II flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ) and FCS Express 4 Flow software (De Novo Software, Glendale, CA). Briefly, cells were collected, washed with cold PBS, and resuspended in 100–150 μL of binding buffer. Cells were stained with 5–7.5 μL of annexin V and 1–1.5 μL of 100 μg/mL PI at room temperature for 15 min and then diluted with 250–400 μL of binding buffer. The intensities of fluorescent annexin V and PI stained cells were quantified by flow cytometry at 450 nm, 530 nm and 695 nm, respectively. The percentages of cells undergoing apoptosis were determined by dual color analysis, which allowed us to distinguish three subsets of cells: viable live cells (annexin V-negative and PI-negative), early apoptotic cells (annexin V-positive and PI-negative), and late apoptotic or necrotic cells (annexin V-positive and PI-positive) [47 (link),48 (link)].