Imagestreamx

To sort the MICs, the MIC-enriched samples were submitted to flow cytometry. Sorting by flow cytometry allowed the separation of the small, diploid MICs from the highly polyploid MAC and the bacteria abundant in Paramecium cultures. P. aurelia MICs were sorted based on the SSC, FSC, DAPI (DNA staining), and GFP signals. P. caudatum MICs, which are bigger than aurelia MICs, could be sorted based on their SSC, FSC, and DAPI signals, without the use of an MIC-specific GFP fluorophore. Quality control was performed by flow cell imaging, using the ImageStreamX (Amnis/Merck Millipore, France) imaging flow cytometer, as previously described [5 (link)]. The MICs represented >99% of the sorted sample, except for P. sonneborni (97%). An example of sorting is shown in S1 Fig.

LNs from C57BL/6 Foxp3-GFP were harvested and fixed immediately in 4% paraformaldehyde. Then, cells were labeled with anti-CD4 and anti-CD44 Abs, permeabilized, and stained for Foxo1 and H3 as described above. GFP staining was used to identify CD4R cells. H3 was used to stain the nucleus. Cells were acquired with ImageStreamX (Amnis; EMD Millipore) equipped with four lasers (375 nm (70 mW), 488 nm (200 mW), 561 nm (200 mW), and 642 nm (150 mW)). Laser intensities were set to maximal values that do not saturate the camera. Cells were gated for single cells using the area and aspect ratio features, and for focused cells using the gradient root mean square feature. Data were analyzed with IDEAS 6.2 software. Foxo1 mean fluorescence intensities were calculated within masks created on H3 (nucleus), CD4 (Cells), and cytoplasm (Cells—nucleus combined mask).

The cells were stained with different antibodies, as listed in Table S2. FACS analysis was performed according to routine protocols using a FACS AriaIII flow cytometer (BD Immunocytometry Systems). The data were analyzed using FlowJo vX.0.6 software (FlowJo, LLC, Ashland, OR, USA). Dead cells were excluded by 7‐AAD staining. To validate the engulfment of USL by tumor cells, multispectral imaging FACS was performed to detect rhodamine‐labeled USL in a single cell using ImageStreamX (Merck, Darmstadt, Germany). In some cases, the Annexin V Apoptosis Detection Kit (eBioscience, Waltham, MA) was used to detect tumor cell apoptosis. FITC‐Annexin V was added to a single‐cell suspension and incubated for 15 min at RT, followed by propidium iodide staining. The cells were then washed with 1× annexin‐binding buffer and gently mixed for further analysis by flow cytometry.

LNs cells of C57BL/6 mice were harvested and fixed in 4% paraformaldehyde, immediately or after 30 min of resting or stimulation with 200 nM of Thapsigargin in RPMI 1640 Glutamax (Gibco). Cells were washed in 1% FCS and 0.1% NaN₃ in PBS and incubated in glycine (0.1M) for 10 min. Cell surface was stained with biotinylated anti-Ly-6C (AL-21), BV 510-conjugated anti-CD4 (RM4-5), PE-conjugated anti-CD25 (PC61), anti-TCRγδ (GL3), anti-CD8.β2 (53–5.8), anti-NK-1.1 (PK136), anti-CD11b (M1/70), PE-Cy7-conjugated anti-CD44 (IM7) and PerCp-Cy5.5-conjugated streptavidin, all from BD Biosciences. Intracellular stainings were performed using Foxp3 Staining kit (eBioscience) and Alexa 448-conjugated anti-NFAT1 (D43B1; Cell Signaling, Leiden, The Netherlands) or anti-NFAT2 (7A6; BioLegend) and APC-conjugated anti-Foxp3 (FJK-165; eBioscience) Abs were used. Ly-6C^- and Ly-6C⁺ CD4 T_N cells were sorted as CD4-BV510⁺ Lineage-PE^- CD44^-/lo Foxp3-APC^- Ly-6C^+/- cells using a FACS-ARIA3 flow cytometer (BD Biosciences). After sort, DRAQ5 (Cell Signaling) was used to stain nuclei. Cells were acquired with ImageStreamX (Amnis; EMD Millipore) and analyzed with IDEAS software. NFAT1 and NFAT2 nuclear localization was calculated as the similarity score between NFAT and DRAQ5 intensities.

For Imagestream analysis of particle internalization, particles were synthesized to incorporate calcein, PPD, and PGN as previously described. A 100 μL of fluorescently tagged particles were added to 10⁶ cells/mL and incubated for 3 h, before washing and re-suspending cells in ice cold PBS at 10⁷ cells in 100 μL. Cells were then stained for 20 min on ice in the dark for the surface marker CD14 PerCP Cy5.5 (BD Biosciences, UK), washed, re-suspended in a small volume of PBS, and filtered before acquisition on an ImagestreamX using INSPIRE V4.1.501.0 software (Amnis-Merck Millipore, USA), acquiring a minimum of 50,000 events per sample. A stained particle control was incubated with cells and acquired in addition to unstained particle/cell negative controls and single surface marker controls required for the generation of a compensation matrix and analysis using IDEAS V6.0 software (Amnis-Merck Millipore, USA).

The following antibodies coupled to different fluorochromes were used for the flow cytometry: anti-CD11c (N418), -CD11b (M1/70), -CD8α (53-6.7), -BST-2 (PDCA-1 eBio129c), -CD80 (16-10A1), -CD86 (GL1), and -CD69 (H1.2F3) from eBiosciences (San Diego, CA, USA); -CD19 (ID3), -H2-K^b (AF6-88-5), -I-A^b (AF6-120.1), and -PDC-TREM (4A6) from Biolegend; and -CD40 (HM40-3) from BD Pharmingen. SIINFEKEL/H-2K^b/PE MHC dextramers (Immudex, Copenhagen, Denmark) were used to identify the OVA-specific CD8⁺ T cells. The control isotypes were purchased from the corresponding providers. Cells were acquired on a Fortessa analyzer (BD). The data were analyzed using the FlowJo Software (Tree Star Inc. Stanford, USA).
To visualize PKH67⁺ apoptotic cells in pDCs, cells were stained with Abs and digital imaging was performed on an imaging flow cytometer (ImageStreamX; Merck). At least, 10⁴ pDCs were imaged for each sample and analyzed using manufacturer’s software (IDEAS).