Macsquantify software

Bacteria from over-night cultures were washed 2x with 1 ml sterile-filtered PBS and diluted to 1x10⁶ CFU/ml in PBS. Flow cytometry was performed using a MACSQuant VYB (Miltenyi Biotec), measuring 100,000 events. YFP was excited with a blue laser (488 nm; bandpass filter 525/50 channel B1), dsRed was excited with a yellow laser (561 nm; bandpass filter 615/20 nm channel Y2). The MACSQuantifyTM Software (Miltenyi Biotec) was used to aquire the data and the FlowJo Software (FlowJo, LLC) for analysis.

Cells were incubated for 30 min with 70 μg/mL cycloheximide (prepared in DMSO), further diluted to reach a cell count between 0.5 × 10⁶ and 1.5 × 10⁶ cells/mL and then immediately injected into a flow cytometer MACSQuant VYB (Miltenyi Biotec, Germany). Regions were determined as a function of the mCitrine and mKate2 fluorescence. Optimal laser and filter setups for the two dyes were as follows: 488 nm laser and 525/25 Band Pass B1-filter for mCitrine, and 561 nm laser and 615/10 Band Pass Y2-filter for mKate2. The expression profile of mKate2 was measured for each sample by the MACSQuant VYB flow cytometer with the MACSQuantify TM Software (Miltenyi Biotec, Germany). A filter was applied on FSC-A/SSC-A to select homogeneous cells regarding size, shape, and cellular complexity. The mean fluorescence value of mKate2 and mCitrine was calculated and exported.

Immunologic parameters were evaluated in the Cell Technology Laboratory of the N.I. Pirogov Russian National Research Medical University 1 week before, 2 weeks, and 8 weeks after immunotherapy during control examinations. The expression of superficial markers was evaluated according to the protocol developed by Miltenyi Biotec GmbH, Germany (manufacturer of antibody kits). The kit contained the following antibodies: FITC-labeled CD3, clone SK7; PE-labeled CD8, clone SK1; CD45 labeled with PerCP, clone 2D1 (HLe-1); APC-labeled CD4, clone SK3; PE-labeled CD16, clone B73.1; PE-labeled CD56, clone NCAM 16.2; and APC-labeled CD19, clone SJ25C1. For intracellular staining of lymphocytes by FoxP3 antibodies, the Treg Detection Kit (Miltenyi Biotec GmbH, Germany) was used. Measurements were performed using a flow cytometer (Miltenyi Biotec GmbH, Germany). The percentage of cells positive for specific markers was calculated using the MACSQuantify (TM) Software (Miltenyi Biotec GmbH, Germany).

The ability of the NCMs to induce changes in the surface receptors of macrophages was evaluated by flow cytometry. The ability of the M0 macrophages to develop an anti-inflammatory profile, and the capacity of the M1 polarized macrophages to revert to an anti-inflammatory phenotype was analyzed. Cells were incubated with NCMs at 5 and 10 NCM/cell ratios for 48 h at 37 °C and 5% CO₂. Next, cells were washed with PBS + 2 mM EDTA + 0.5% BSA, detached with TripLe™ and counted. 100,000 M0 or M1 were added to flow cytometry tubes. After Fc receptor blockade, cells were stained with anti CD14, CD64, CD83 and CD206 fluorescent antibodies, washed and analyzed by multicolor flow cytometry (MACSQuant 10, Miltenyi Biotec, Madrid, Spain). Macrophages were gated according to their forward- and side-scatter characteristics and data were analyzed by MACSQuantify software (Miltenyi Biotec, Bergisch Gladbach, Germany).

Cells were harvested by trypsinization, fixed with 70% ethanol, briefly washed with PBS/0.2% Triton X-100 and subsequently incubated with PBS/0.2% Triton X-100/DAPI (4′,6-Diamidin-2-phenylindol, 1 µg/ml) for 30 min at room temperature in the dark. Cells were washed once with PBS/0.2% Triton X-100 and flow cytometric analysis was performed using a MACSQuant10 with MACSQuantify Software (Miltenyi Biotec). The portion of cells in the respective cell cycle phases was calculated using ModFit LT™ software (Verity Software House).

Cell numbers from the peritoneal cavity and spleen were determined by the flow cytometry absolute counting system with a MACSQuant^® Analyzer. Cells (0.5 × 10⁶) were incubated in HBSS, 0.5% BSA, 10 mM HEPES containing mouse FcR Blocking Reagent, following the manufacturer’s instructions. Cell viability was assessed using Viobility™ 488/520 Fixable Dye. Incubation with antibodies was performed at 4 °C for 30 min in the dark. The antibodies used were Ly-6G VioBlue^®, CD45 VioGreen^®, CD11c PE-Vio^® 770, and F4/80 APC. Acquisition was performed using a MACSQuant^® Analyzer flow cytometer using MACS Quantify software (Miltenyi Biotec). The flow cytometry data were analyzed using FlowJo™ Tree Star software (Ashland, OR, USA). The concentration of the different cytokines in the intraperitoneal fluid was determined using an ELISA kit from Bio-Techne (Minneapolis, MN, USA) for IL6, IL1β, and TNFα, following the manufacturer’s instructions.