Cytofix cytoperm permeabilization kit

Cell phenotype was performed on lymph nodes, splenic and bone marrow lymphoid populations by four-color fluorescence analysis according to standard protocols. The following antibodies and reagents were used: PerCP, PE, or APC anti–mouse B220, CD3, CD4, CD8, CD19, CD21, CD23, CD44, CD69, CD86, I-A/I-E, CD25, CD138, and IgM (all from BD Biosciences). Propidium iodide was used for live-dead discrimination. For proliferation analysis, cells were permeabilized after extracellular staining and fixed with the cytofix/cytoperm permeabilization kit (BD Biosciences), then stained with the PerCP Cy5.5 anti-Ki67 (BD Biosciences). Cells were analyzed using a FACSCalibur. We then analyzed data with FlowJo software (Treestar). Bone marrow and splenic B-cell subsets (Supplementary Fig. S4) on the one hand, and PB and undifferentiated B cells after 4 days of culture with LPS (Fig. 6) on the other hand, were sorted by flow cytometry using a FACS Aria cell sorter (BD Biosciences), after staining with the antibodies indicated in the legend of the figures.

PBMCs will be cultured in 48-well culture plates at 0.5 to 1 × 10⁶ cells/mL per well and will be stimulated with 5 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (both from Sigma Aldrich, Oakville, Ontario, Canada) for two hours at 37°C. A total of 2 μg/ml brefeldin A (Sigma Aldrich, Oakville, Ontario, Canada) will then be added to block cytokine secretion and cells will be cultured for 18 hours at 37°C. PBMCs will be surface stained followed by fixation and permeabilization using the Cytofix/Cytoperm Permeabilization kit (BD Bioscience, Mississauga, Ontario, Canada) for intracellular staining for IL-17A and IFN-ɣ (positive control). Treg cells will be defined by flow cytometry as being CD3+ CD4+ CD25^high CD127^low FoxP3^high and Th17 cells as being CD3+ CD4+ IL-17A+ upon PMA and ionomycin stimulation [27 (link)].

To identify the cellular phenotype of platelets, expression of surface molecules was examined by flow cytometry. For gating purposes, the platelet population was verified using anti-CD41a-VioBlue (Clone REA386; Miltenyi Biotec, Auburn, CA, USA) monoclonal antibody (mAb) staining. Antibodies used to probe surface molecules included anti-P-selectin-FITC (Clone CLBThromb/6; Beckman Coulter, Westbrook, ME, USA), anti-TLR2-Alexa Fluor 488 (Clone T2.5; Abcam), anti-TLR4-APC (Clone HTA125; Abcam) and anti-TLR9-FITC (Clone 5G5; Abcam) mAbs. Isotype-matched control IgGs for each mAb were used to set negative populations. Platelets were analyzed on a MACS Quant Analyzer (Miltenyi Biotec) and data were analyzed using FlowJo v.7.6.4 software (Tree Star, Ashland, OR, USA). Details of the gating strategies of platelets are shown in Fig. S1. As shown in Fig. S1, TLR9 is found in intracellular domains, though it has been reported that TLR9 could migrate to the outer membrane after activation^{34 (link)}. TLR9 expression was measured after permeabilization of the cells using cytofix/cytoperm permeabilization kit (BD Biosciences, San Jose, CA, USA).