Aqua live dead fluorescent dye

For in vitro experiments, BMDCs were generated as described previously^{51 (link)}. Cells were plated on day 10 at concentrations of 1 × 10⁶ cells/ml and stimulated 2 h later with decreasing doses of M9 OMVs, 5 ng/ml E. coli LPS (Sigma) or left unstimulated for 24 h. For the analysis of surface marker expression, cells were incubated with AQUA LIVE/DEAD fluorescent dye (0.5 μl) (Invitrogen) for 30 min. Cells were subsequently stained with anti-CD11c (PE-Cy7, eBioscience), anti-CD40 (APC, eBioscience), and anti-CD80 (PerCP-Cy5.5, BD Biosciences) then measured by flow cytometry as described above. The levels of IL-12p70, IL-18, and IL-6 cytokines in culture supernatants were measured by multiplex assay (BioRad).
For in vivo studies, C57Bl/6 mice were injected intraperitoneally with 100 μl saline control or 10 μg OMVs that were pre-labeled with CFSE (CellTrace, Life Technologies) according to the manufacturer’s instructions. After 6 h, cellular exudates were obtained by peritoneal lavage. Cells were stained with anti-CD11c, anti-CD40 (PE), anti-CD80 (PE-CF594), anti-CD86 (BV605), anti-MHC class I (APC), anti-MHC class II (PerCP Cy5.5), and analyzed by flow cytometry. The levels of IL-12p70, IL-18, and IL-6 cytokines in lavage fluid were measured by multiplex assay (BioRad).

Blood was collected from tail vein or by cardiac puncture and added to FACS buffer (PBS/1% FBS/2 mM EDTA). RBCs were lysed using 0.2% NaCl, followed by 1.6% NaCl. Kidneys were harvested following perfusion with PBS. Briefly, kidney homogenates were digested in HBSS with 2 mg/ml collagenase type I (Worthington, Lakewood, NJ) and DNase-1 (MP Biochemicals, Solon, OH) for 30 min at 37°C. Kidney cell suspensions were passed through a 40-μm filter and overlaid on Lympholyte-M (CEDARLANE, Burlington, NC). Cells were stained with an Aqua Live/Dead fluorescent dye (Molecular Probes, Eugene OR) and blocked with rat anti-mouse CD16/32 (eBioscience). Cells were stained with the following Abs (from BD Biosciences unless noted): Ly6G-PE or -FITC (clone 1A8), Ly6C-PerCP-Cy5.5 (clone AL-21), CD11b-BV421 or -allophycocyanin (clone M1/70), CD45-FITC (clone 30-F11), and TNF-α–allophycocyanin (clone MP6-XT22; eBioscience). Samples were acquired on a FACS ARIA II or Fortessa (BD Biosciences) and analyzed with FlowJo software (TreeStar).
For histology, kidneys were fixed in 10% formalin, embedded in paraffin, and processed for staining with H&E or periodic acid–Schiff (PAS) with Light Green SF Yellowish counterstain for collagen (Sigma). Images were taken with an EVOS FL Auto microscope (Life Technologies).