Attune nxt software

Cancer cell lines and cancer SC (CSC)-like cells were collected and prepared according to the manufacturer’s instructions. The following monoclonal antibodies were obtained from BD Biosciences (Los Angeles, CA, USA): phycoerythrin (PE) mouse anti-human CD44, and allophycocyanine (APC) mouse anti-human CD133, and their immunoglobulin G (IgG) controls. Reacted cells were analyzed using an Attune^TM NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific), and each flow cytometer measurement used AttuneTM NxT software (Thermo Fisher Scientific) for data processing. Individual CD44+CD133⁻ sphere and CD44+CD133+ sphere populations were sorted using a FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA) from CD44+ spheres obtained from the sphere-forming assay. Three independent experiments were performed for the statistical analysis.

At 24, 48, and 72 h p.i., virus-draining popliteal LNs and non-draining brachial LNs were harvested from recipient mice and homogenized using a Corning™ Falcon™ tube with Cell Strainer Snap Cap (5 ml). 2–5 × 10⁶ cells/well were plated on 96-well V-bottom plates and Fc receptors were blocked with 2.4G2 mAb for 20–30 min at 4°C. Cells were stained with fluorochrome—conjugated mAbs CD8 (53–6.7), CD4 (GK1.5), and CD69 (H1.2F3) from BioLegend (San Diego, CA) in 50–100 μl FACS buffer for 20 min at 4°C. Alternatively, cells were stained with biotinylated mAb against CD25 (PC61) followed by secondary staining with fluorochrome-conjugated streptavidin for 20 min at 4°C. For the proliferation assay at 72 and 120 h p.i., cells were stained for with fluorochrome-conjugated mAbs against CD8 (53–6.7) and CD4 (GK1.5). For DC activation analysis at 24 h p.i., cells were stained with fluorochrome—conjugated against mAbs CD11c (N418), MHC class II (M5/114.15.2), CD80 (16–10A1), and CD86 (GL-1) from BioLegend (San Diego, CA) in 30 μl FACS buffer for 30 min at 4°C. Zombie Red™ (BioLegend) was used as viability dye. After staining, the fixation was performed with 1% PFA for 20 min at 4°C. Samples were analyzed using AttuneTM NxT Flow Cytometer and AttuneTM NxT Software (ThermoFisher), FlowJoTM 10 software (Treestar) and GraphPad Prism.

The primary RipTag2 cells were plated with WT or MK2 KO BMMs in a ratio of 1:2. Some WT or MK2 KO BMMs were activated with 1µg/mL of LPS (Enzo Life Sciences, Farmingdale, NY, USA). Both non-activated and activated WT or MK2 KO BMMs werecultured with primary RipTag2 cells and incubated for 24 h. Supernatants from co-cultures were collected for multiplex analysis. Cells were stained with F4/80 (cat. 14-4801-82, ThermoFisher Scientific, Waltham, MA, USA) and Annexin V Ready Flow Conjugate for Apoptosis Detection using Annexin Binding Buffer (cat. R37174 and cat. V13246, ThermoFisher Scientific, Waltham, MA, USA). Flow cytometry analysis was performed using an Attune^TM NxT Flow Cytometer and analyzed with Attune^TM NxT Software (ThermoFisher Scientific, Waltham, MA, USA). Macrophages were gated using F4/80 marker and the percent of annexin V⁺ cells in the population of tumor cells was calculated.

To investigate apoptosis in mel P cells, we used Annexin V for detection of the phosphatidylserine externalization—one of the early apoptosis markers. Briefly, cells were seeded on 6-well plates (2 × 10⁵ cells/well) and incubated with 10 μM of mambalgin-2 for 72 h. After incubation, the cells were detached by the Versene solution and washed in the annexin-binding buffer (V13246, Thermo Fisher Scientific). Then, the cells were incubated with Annexin V conjugated to Alexa488 (A13201, Thermo Fisher Scientific) for 20 min, washed by the annexin-binding buffer, and were analyzed using the Attune NxT flow cytometer (Life Technologies). The data were analyzed using the Attune NxT Software (Life technologies).

Cells were washed and incubated with a PE-conjugated anti-CD4 antibody (clone VIT4; Miltenyi; dilution 1:200) for 30 min at 4 °C. Cells were fixed with 4% PFA for 10 min at room temperature. Cells were washed and stained with a FITC-conjugated anti-Gag antibody (clone KC57; Beckman-Coulter; diluted 1:500 in PBS/BSA 1%/Saponin 0.05%) for 30 min at room temperature. Cells were washed and resuspended in PBS. Data were acquired on an Attune NxT flow cytometer (Life Technologies)using the Attune NxT Software (Life Technologies). Data were analyzed using the FlowJo software (v10.7; BD). The Median Fluorescence Intensity (MFI) of Gag in infected (CD4⁻Gag⁺) cells was calculated and normalized to the condition without antibody.

To investigate apoptosis in A549 and Lewis cells, we used Annexin V for detection of phosphatidylserine externalization—one of the early apoptosis markers. Briefly, cells were seeded in six-well culture plates (2 × 10⁵ cells per well) at pH 5.5, grown for 24 h, and incubated with 1 μM mambalgin-2 for 72 h without the media change. After incubation, the cells were detached using the Versene solution and washed with annexin-binding buffer (V13246, Thermo Fisher Scientific). Then, the cells were incubated with Annexin V conjugated to Alexa 488 (A13201, Thermo Fisher Scientific) for 20 min, washed with the annexin-binding buffer, and analyzed using the Attune NxT flow cytometer (Life Technologies). The data were analyzed using the Attune NxT Software (Life Technologies).