Facs calibur 2 flow cytometer

Phagocytosis assays were carried out as previously described (11 (link), 17 (link)). Briefly, fresh sheep red blood cells (RBCs) (Thermo Scientific) labelled with 2 μM DDAO-AM (Invitrogen) were incubated with sub-agglutinating concentrations of polyclonal rabbit IgM anti-sheep RBC cells (MyBioSource) and later treated with 10% C5-deficient human serum (Sigma) for complement opsonization. Differentiated HL-60 cells starved for 3 h in serum-free RPMI, were treated with 320 nM LPS (Sigma) for 30 minutes or 1 mM MnCl₂ for 5 minutes. Cells were incubated with complement-opsonized RBCs (C3-RBC) or unopsonized RBCs as negative control, for 30 min at 37°C in a 1:10 ratio, and unbound RBCs were washed thrice with ice-cold PBS. To determine particle internalization, cell-bound RBCs were exposed to a 30 s hypotonic shock with distilled H₂O, and isotonicity restored with an equal volume of twice-concentrated PBS. Cells were analyzed in a BD FACSCalibur II flow cytometer (BD Biosciences), using the FlowJo package (BD Biosciences) and expressed as Association Index (AI), indicating the number of cells with attached and engulfed particles, or Phagocytic Index (PI) indicating cells with internalized particles (11 (link)). These indexes are all normalized with respect to the AI for unstimulated control cells.

Apoptosis was analyzed by the FITC-Annexin V plus PI staining method. The transfected cells were harvested by trypsinization. After washing with phosphate buffered saline (PBS) containing 1% FBS, the cells were stained with 5 μl FITC-Annexin V and 2 μg/ml PI. The fluorescence intensity in the cells was assessed using BD FACSCalibur II flow cytometer and the CellQuest software (BD Biosciences, San Jose, CA). In each analysis, 20,000 gated events were recorded.

HepG2 cells were exposed to 300 μM oleic acid for 24 h after transfection with AhR plasmid for 48 h, and the cells were harvested by trypsinization. After washing with PBS, the cells were stained with 30 nM Nile Red or 0.05 μg/ml Rhodamine 123 for 30 min in cell culture media containing 20% FBS. In each analysis, 20,000 gated events were recorded. The fluorescence intensity in the cells was assessed using the FACS Calibur II flow cytometer and the CellQuest^TM Pro software (version 6.0, BD Biosciences, San Jose, CA, USA). A schematic representation of the gating strategy can be found in Supplementary Fig. 9.

For detection of cytokine levels blood was collected 12 hours after sepsis induction into heparinized tubes, through cardiac puncture. Plasma was separated immediately from whole blood by centrifuging at 3000 r.p.m for 15 minutes at 4′C. Multiple soluble cytokines IL-1α, IL-2, IL-4, TNF-α, IL-10 were simultaneously measured by flow cytometry using the cytometric bead array (CBA) Rat Inflammation kit (BD Biosciences, USA). Acquisition was performed using a FACS Calibur II flow cytometer (BD Biosciences, USA). Quantitative results were generated using the FCAP array software. For the measurement of cytokines TNF-α, IL-6, IL-1β and IFN-γ in the organ homogenates, ELISA kits (Bioassay Technology Laboratory, China) were used. Organ homogenates were collected by homogenizing 100 mg of tissue in 1 ml sterile 1X PBS followed by two freeze thaw cycles to break the cell membranes. The homogenates were centrifuged for 5 minutes at 5000 × g at 4′C and the supernatant was aliquoted and stored at −20 °C until analysis.