Macsquant device

Cells were incubated with a phycoerythrin (PE)-labelled HLA-A*02:01 PSF1_79–87 peptide-binding HLA tetramer (PSF1 tetramer) (Medical & Biological Laboratories, Nagoya, Japan) and then stained with an allophycocyanin (APC)-labelled anti-human CD8 monoclonal antibody (BD Biosciences). The cells were analysed by a MACSQuant device (Miltenyi Biotec). HLA-A*02:01-restricted PSF1_79–87 CTLs were defined as CD8⁺/PSF1-tetramer⁺ T cells^{48 (link)}.

Single cell suspensions from footpads, draining lymph nodes or spleens were surface stained for phenotypic analysis. Cells were incubated for 30 minutes with appropriate amounts of antibodies in the presence of Fc receptor-blocking agent (Fc block from BD Bioscience), after which cells were washed and stained with a viability dye (eBioscience). Antibodies used were directed against mouse CD45 (GL2), CD11c (HL3), CD40 (3/23), CD3 (145-2CII), CD4 (RM4-5), CD8 (53–6.7), CD25 (PC65), CD19 (ID3), CD11b (M1/70), Ly6G (1A8-Ly6G), Ly6C (HK1.4) and NK1.1 (PK136) from BD Biosciences and eBioscience. For intracellular staining, surface-labeled cells were fixed and permeabilized with the Inside Stain Kit from Miltenyi Biotec for IFNγ labelling with an anti-IFNγ (XMG1.2) or fixed and permeabilized with the transcription factor permeabilization kit from eBioscience and stained for foxp3 (MF23 from BD Biosciences). All controls were stained with the respective isotypes. Flow cytometric data were acquired on a MACSQuant device (Miltenyi Biotec) and analysed using FlowJo software (TreeStar).

ravvia promoter sequence fused to gfp by amplification of Pravvia-gfp from pZE1-gfp (63 (link)) using primers ZIP537/ZIP200. The fragment was cloned into pTOPO-TA cloning vector. The Pravvia-gfp fragment was then extracted using EcoRI and cloned into the low copy plasmid pSC101. The plasmid was introduced into desired strains, and fluorescence was measured on indicated conditions, by counting 100,000 cells on the Miltenyi MACSquant device.

Experiments were performed as described ^{60 (link)}. Briefly, overnight cultures were diluted 100 fold MOPS Rich medium. When the bacterial strains reached an OD of 0.25, cells were incubated them with 0.4 μM of Cy5 labeled Neomycin for 15’at 37°C. Then, 20 μl of the incubated culture were used for flow cytometry, diluting them in 200 μl of PBS before reading. Flow cytometry experiments were performed as previously described ^{61 (link)}. For each experiment, 50 000 events were counted on the Miltenyi MACSquant device. The Y3 fluorescence channel was then used to measure fluorescence intensity.

SOS induction measurements by flow cytometry was performed as previously described [17 (link),18 (link),25 (link)]. Briefly, overnight cultures were diluted 100-fold in MH or MH supplemented with subMIC tobramycin (0.02 μg/mL), subMIC ciprofloxacin (0.005 μg/mL) and/or AI-2 (10 μM) and were incubated overnight at 37 °C. Fluorescence was then measured in 100,000 cells on the Miltenyi MACSquant device. The fluorescence values in each condition were normalized to the fluorescence values obtained in MH.

CB samples collected in heparin lithium vacutainers were processed to isolate peripheral blood mononuclear cells (PBMC) by gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, MO, USA) and cell subsets were obtained by immuno-magnetic cell sorting (RoboSep™-S, STEMCELL™ Technologies, Canada). B cells, T cells, granulocytes and hematopoietic progenitor cells (HPC) were respectively sorted with EasySep^® human whole blood CD19, CD3, CD66b positive selection kit and human cord blood CD34 positive selection kit following manufacturer’s recommendations.
Fractions were checked for purity by flow cytometry with the MACSQuant^® device (Miltenyi Biotec, Germany) using CD20-VioBlue^®; CD4-(VIT4)-FITC; CD8-PE and CD66abce-APC fluorescent antibodies, following manufacturer’s recommendations. Cell fractions with purity higher than 95% were kept for microchimerism analysis.

Quantification of fluorescent neomycin uptake was performed as described (29 (link)). Neo-Cy5 is an aminoglycoside coupled to the fluorophore Cy5 and has been shown to be active against Gram-negative bacteria (31 (link), 55 ). Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ-rich defined medium). When the bacterial strains reached an OD 600 nm of ∼0.25, they were incubated with 0.4 µM of Cy5-labeled neomycin for 15 min at 37°C. Ten microliters of the incubated culture was then used for flow cytometry, diluting them in 250 µL of PBS before reading fluorescence. WT V. cholerae was incubated simultaneously without Neo-Cy5 as a negative control. Flow cytometry experiments were performed as described (56 (link)) and repeated at least three times. For each experiment, 100,000 events were counted on the Miltenyi MACSquant device.