Gallios cytofluorometer

Flow cytometry was performed using a Gallios cytofluorometer™ (Beckman Coulter, Fullerton, CA, USA) in order to analyze cell viability, apoptotic and necrotic cell numbers, intracellular reactive oxygen species (ROS) generation and cellular thiol content of primary human cells. The experimental set up and analyses were performed according to the protocol used in a study of Daum et al. [54 (link)].

Neutrophils were freshly isolated from EDTA-blood from healthy donors by standard density gradient centrifugation using Ficoll (Lymphoflot, BioRad) followed by two cycles of hypotonic erythrocyte lysis with sterile water. Purified neutrophils were kept in PBS supplemented with 2 mM EDTA and 2% FCS at a concentration of 2 × 10⁶ cells/ml and incubated with heat aggregated IgA at the indicated concentrations for 20 min at 4 °C. After washing, neutrophils were incubated with 1.25 µg/ml fluorescein isothiocyanate (FITC)-labeled goat F(ab)₂ anti-human IgA (#2052-02; Southern Biotech) for 20 min at 4 °C.
Polystyrene beads (size 1 µm; Color Y; Estapor, Merck) were incubated with 100 µg/ml IgA in PBS for 1 h at room temperature and constant agitation. After washing, the beads were incubated with 1.25 µg/ml FITC-labeled goat F(ab)₂ anti-human IgA (Southern Biotech) in PBS supplemented with 2 mM EDTA and 2% FCS for 20 min at 4 °C.
Flow cytometry was performed on a Gallios cytofluorometer and evaluated using Kaluza Analysis 2.1 software (both Beckman Coulter). Cell or bead aggregates, dead cells, and cell debris were excluded. Delta mean fluorescence intensity (ΔMFI) values were calculated by subtracting the MFI of neutrophils or beads stained with secondary antibody only.

Viability of HUVECs was measured using Gallios cytofluorometer™ (Beckman Coulter, Fullerton, CA, USA). Briefly, 10 × 10⁴ cells/well were seeded into 12-well plates and samples were incubated at the respective temperature (37 °C, 25 °C or 4 °C) for 3 h or 24 h. Following the incubation process, cells were harvested and 50 µl of single cell suspensions were incubated with 250 µl of staining solution for 30 min at 37 °C. For 1 ml staining solution, 10 μg Hoechst 33342, 25 ng AxV-FITC and 66.6 ng PI (propidium iodide) were added to Ringer solution. Samples were analyzed using flow cytometry with the following laser setting: FITC fluorescence was detected with the FL1 sensor (525/38 nm band pass filter, BP); Hoechst 33342 fluorescence was detected at 405 nm using the FL9 sensor (430/40 nm BP) and the PI fluorescence was detected with the FL3 sensor (620/30 nm BP). Electronic compensation was used to eliminate fluorescence bleed-through. Data analysis was carried out with the Kaluza software Version 1.2 (Beckman Coulter).

For ex vivo phagocytosis experiments, anticoagulated fresh whole blood was incubated at 37 °C with UMA. Erythrocytes were subsequently removed by hypotonic lysis. Leukocytes were stained with anti-CD16 (cat. no. B-E16-FITC, Abcam) and anti-CD14 (cat. no. 61D3-PE, eBioscience) and analyzed by flow cytometry. For in vitro phagocytosis experiments, purified neutrophils (2 × 10⁶ cells/ml) were stimulated with 50 pg of UMA per cell in the presence of 10% autologous serum. Serum had been pre-treated as indicated. The phagocytic indices (PhIx) for UMA were calculated by multiplying the percentage of cells with increased side scatter with the mean side scatter value. Where indicated, phagocytosis was confirmed by exposing neutrophils to UMA in the presence of 100 nM pHrodo Red SE (Life Technologies) and subsequently analyzing cell-borne fluorescence by flow cytometry. Flow cytometry and data analyses were performed employing the Gallios cytofluorometer and the Kaluza software, respectively (Beckman Coulter).

Monoclonal antibodies directly conjugated with fluorescence and isotype controls were used for surface or intracellular staining with optimal concentration for flow cytometry analysis. The antibodies are listed in Supplementary Table 2. Before intracellular cytokine staining, the cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml, Wako) and ionomycin (750 ng/ml, Wako) plus Golgi Plug (1 μl/ml, BD Biosciences) for 4–5 h. The cells were surface-stained, fixed, permeabilized, and stained for intracellular and intranuclear targets using BD Cytofix/Cytoperm (BD Biosciences) and True-Nuclear Transcription Factor Buffer Set (BioLegend), respectively, according to the manufacturers’ protocols. Analyses of the samples were performed on either a Gallios cytofluorometer, CyAn ADP analyzer (Beckman Coulter, CA, USA) or an LSR-II (BD Biosciences), and the data were analyzed using Kaluza analysis software (Beckman Coulter) or FlowJo (Tree Star, OR, USA). The gating strategies are shown in Supplementary Fig. 3.