Lrs fortessa cytometer

Single cell suspensions from mouse spleens were obtained via mechanical disruption through 40 µm cell strainer (Falcon) and red blood cell lysis through 1xRBC Lysis Buffer (eBioscience). Cells were incubated on ice for 30 min with the combination of primary fluorochrome-conjugated antibodies or primary unconjugated biotin antibodies followed by conjugated streptavidin (complete list in Supplementary Table S1) in PBS supplemented with 0.5% BSA. Viability markers were also used according to manufacturer’s instructions. For intracellular isotypes staining, cells were first stained with surface antibodies then fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience). Cells were washed, resuspended in PBS containing 2% FCS and acquired on BD LRS Fortessa cytometer (Beckton Dickinson). In cell sorting experiments, total spleen B cells were first purified with the Pan B Cell Isolation Kit II mouse (Miltenyi) following manufacturer’s instructions then sorted into GL7⁺PNA⁺EYFP⁺ GC or GL7^-PNA^-EYFP⁺ memory cells with a FACSAria (Beckton Dickinson). Analyses were performed with FlowJo (Tree Star Inc.) software.

The native and genetically modified MDA-MB-231 (3 × 10⁵) and T47D cells (5 × 10⁵) were grown in 6-well plates (Greiner BioOne). After 24, 48 and 72 h, the cells were fixed in 70% cold ethanol (v/v) and stained with propidium iodide (PI) solution according to the manufacturer’s protocol (Life Technologies, CA, USA). After incubation at 37 °C in the dark for 30 min, PI fluorescence was measured in the FL-2 channel of the BD LRS-Fortessa cytometer (Becton Dickinson). The data from minimum 20,000 events per sample were collected, processed and analyzed using ModFit LT™ version 5.0 (Verity Software House, Inc., USA). The experiments were repeated 3 times.

After differentiation in monoculture or co-culture in vitro, Mphs and mDCs were harvested using StemPro^® Accutase^® (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 37 °C. The phagocytic potential of polarized Mph or mDC was assessed using pHrodo™ red-conjugated yeast and bacterial particles (Zymosan A, E. coli, or Staphylococcus aureus [S. aureus]; Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. These particles can detect the acid pH of the phagosome and fluoresce to indicate that phagocytosis is taking place. Fifty thousand cells were transferred to 96-well Ultra-Low Attachment U-bottom wells (Corning Biosciences, Corning, NY, USA), and cells were rested for 60 min in RPMIc. Lyophilized pHrodo™-conjugated particles were reconstituted in 1 mL of RPMIc per vial prior to use, and particles were sonicated for 5 min at 20% amplitude. pHrodo™ Zymosan or bacterial particles (50 μL) were added to each well, and the cells were incubated for 60 min (37 °C, 5% CO₂). Afterwards, phagocytosis was stopped by placing the samples on ice, and cells were washed prior to fluorescence-activated cell sorting analysis using a LRSFortessa™ cytometer (BD Biosciences, Haryana, India). Negative controls for phagocytosis were cells without pHrodo™ reagent. The results were analyzed using the FlowJo™ software.