Lsr fortessa flow cytometry analyzer

For the assessment of intracellular iNOS expression, a total of 1 × 10⁶ BMDN in RPMI medium were stimulated with 1 µg/ml rmCIRP for 4 h at 37°C in 5% CO₂ cell culture incubator. Following incubation, the cells were stained with APC anti-mouse Ly-6G Ab (clone: 1A8; Biolegend) and FITC-anti-mouse ICAM-1 Ab (clone: 3E2; BD Biosciences) and then fixed and permeabilized using BD cytofix/cytoperm plus fixation/permeabilization kit (Cat. No.: 554715; BD Biosciences), followed by the intracellular staining with PE-anti-mouse iNOS Ab (Cat. No.: sc-651; Santa Cruz Biotechnology, Santa Cruz, CA). Acquisition was performed on 30,000 events using a BD LSR Fortessa Flow Cytometry Analyzer (BD Biosciences) and data were analyzed by FlowJo software (Tree Star, Ashland, OR).

For flow cytometry analysis of CNC uptake to phagocytic immune cells, mice bearing orthotopic 4T1-tdTomato mammary fat pad allografts were administered with 50 μg of DFO-CNC-Cy5 and NOTA-CNC-Cy5 in 200 μl of 0.5% Kolliphor HS 15–1 × PBS, pH = 7.4 or vehicle intravenously. At 120 min after administration, the mice were sacrificed by CO₂ asphyxiation and perfused transcardially with ice-cold 100 units ml⁻¹ heparin solution in 1 × PBS, and the tumor and spleen were collected. Minced tissues (200 mg for tumor and 100 mg for spleen) were digested with a cocktail containing 4 units ml⁻¹ liberase TH, 40 units ml⁻¹ hyaluronidase, and 40 units ml⁻¹ DNAse at 37 °C for 30 min (spleen) and 60 min (tumor). The cell suspension was filtered through a 70-μm cell strainer and the cells were collected by centrifugation at 400 g at 4 °C for 5 min. The red blood cells in the spleen sample were removed by a treatment with red blood cell lysis buffer (BD Biosciences, San Jose, CA, USA). The cell number in the suspensions was determined on an automated cell counter (Vi-CELL, Beckman Coulter, La Brea, CA, USA) and the concentration adjusted to 10 × 10⁶ cells ml⁻¹. Aliquots of 2 × 10⁶ cells per sample were stained with the appropriate antibodies (see Supplementary Information for dilutions and respective fluorescent labels) and DAPI and analyzed on a BD LSR Fortessa flow cytometry analyzer.

NET contents in the lungs were assessed by flow cytometry.^{33 (link)} Briefly, single-cell suspensions were fixed with 2% of paraformaldehyde for 20 minutes at room temperature, blocked for 30 minutes with 2% of bovine serum albumin (BSA) in PBS at 37°C. Cells were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse anti-MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG. To avoid staining of intracellular chromatin and enzymes, cells were not permeabilized. More than 10 000 events were acquired using a BD LSR Fortessa Flow Cytometry Analyzer (BD Biosciences) and the data were analyzed by FlowJo software (Tree Star, Ashland, OR). In the flow cytometry dot plots, at first the cells were gated based on their size and intracellular granularity by forward and side scatters, respectively. Next, neutrophils were gated based on Ly6G positive staining. In the Ly6G positive population, we then gated the cells based on surface/extracellular MPO and citH3 staining. We chose quadra/rectangular gate to select MPO⁺citH3⁺ in Ly6G⁺ neutrophils to define the NETs⁺ neutrophils. Purified rabbit polyclonal IgG and FITC-mouse IgG1 were used as the isotype controls.

A number of 2×10⁶ BMDN isolated from WT and TLR4^−/− mice in RPMI 1640 medium were stimulated with PBS or rmCIRP (1 μg/ml). After 4 h 37 °C incubation, the cells were washed and resuspended with 1 ml of 0.5% BSA in PBS. LDN was isolated using the Histopaque gradient as described above. LDN were stained with APC anti-mouse Ly-6G Ab (clone 1A8; Biolegend) and PerCP/Cyanine5.5 anti-mouse/human CD11b antibody (clone: M1/70; Biolegend), and accessed by flow cytometry. The frequency of Ly6G⁺CD11b^hi and Ly6G⁺CD11b^lo were assessed as described above. More than 30,000 events were acquired using a BD LSR Fortessa Flow Cytometry Analyzer (BD Biosciences) and the data were analyzed by FlowJo software (Tree Star).