Trucount beads

For phenotypic analysis, animals were sacrificed at 24 hours via CO₂ euthanasia. Tissue harvested for examination included the spleen, MLNs, and Peyer’s patches. Each tissue was processed into single suspensions. The number of cells per milliliter of suspension was obtained via a Nexcelom Auto Cellometer. We plated 2 × 10⁶ cells onto a 96-well plate and stained them for extracellular markers. TruCount Beads (BD Pharmingen) were prepared according to the manufacturer’s instructions and used to determine absolute cell counts.
For cytokine analysis, 2 × 10⁶ splenic cells were plated onto a 96-well plate. Cells were suspended in RPMI 1640 culture medium and incubated for 4 hours at 37°C using PMA (30 ng/mL) and ionomycin (400 ng/mL) with 10 μg/mL Brefeldin A. After incubation, cells were stained with extracellular markers, processed with an intracellular staining kit (BD Biosciences) per manufacturer’s protocol, and stained for intracellular cytokine markers. TruCount Beads (BD Pharmingen) were prepared according to manufacturer’s instructions and used to determine absolute cell counts. Additional data on markers, conjugates, and clones used for flow cytometry are available in Supplemental Table 1. Data were acquired on an LSRII Flow cytometer (BD Biosciences) and subsequently analyzed with FlowJo 10.0.8rl software (Tree Star).

For bacterial abundance, 4 mL of sample were fixed with 200 μL glutaraldehyde (25%) and stored at −20 °C until enumeration within six months from collection. Abundance was determined after staining with SYBR Green I (Invitrogen) and the analysis performed with a flow cytometer (FACSCalibur, Becton Dickinson). Bacterial cell numbers were estimated by manual gating of the bacterial subpopulation in the cytogram of side scatter versus green fluorescence, with a standard deviation <3% between replicate measurements. Yellow-green fluorescent latex beads (Polyscience) and TruCount beads (Becton Dickinson) were used to normalize the counted events to volume71 .

For flow cytometry counts of microalgae and free-living bacteria, 0.05 ml of culture was sampled, diluted at 1:10–1:10,000 and fixed for 15 min in the dark with a final concentration of electron microscopy–grade glutaraldehyde of 0.25% and Pluronic F-68 of 0.01% (Marie et al., 2014 (link)), flash-frozen in liquid nitrogen, and stored at −80°C until the analysis. Cell counts were performed with a BD FacsCanto II Flow Cytometry System [3-laser, 8-color (4-2-2), BD-Biosciences] equipped with a 20-mW 488-nm coherent sapphire solid-state blue laser. Accurate analyzed volumes and subsequent estimations of cell concentrations were calculated using Becton-Dickinson Trucount™ beads. Phytoplankton and bacterial cells were discriminated and enumerated according to their side scatter properties (SSC) for both and red fluorescence (>670 nm) due to chlorophyll pigments or green fluorescence due to SYBR Green I staining of the bacterial DNA [1:10,000 final concentration (Marie et al., 1997 (link))], respectively. Data were acquired using DIVA software provided by BD Biosciences.