Live dead fixable near ir dead cell stain

Popliteal and brachial LNs of the WT and KO mice at steady-state and 1 day after s.c injection of OVA (1 mg/ml) (1:1) (EndoGrade) emulsified in incomplete Freund’s adjuvant (Sigma) into the footpads were collected and digested to obtain single-cell suspensions. Thereafter, the suspensions were depleted from hematopoietic cells using mouse anti-CD45 microbeads (Miltenyi Biotec). The enriched cells were stained with LIVE/DEAD fixable near-IR dead cell stain (Thermo Fisher Scientific) and conjugated primary antibodies against mouse Podoplanin (BioLegend) and CD31 (BioLegend). Live lymphatic endothelial cells (CD45^- CD31⁺ Podoplanin⁺) were sorted into TRIsure (Bioline) using Sony cell sorter, equipped with a 100 µm tip.
Total RNA was extracted from the LECs with RNeasy Plus Micro kit (QIAGEN) and the libraries were prepared using SMART-Seq v4 Ultra Low Input RNA Kit (Takara) and Illumina Nextera XT DNA Library Preparation protocols (Illumina). Sequencing was performed with the Illumina HiSeq 3000 instrument using single-end sequencing chemistry with 50-bp read length at the Finnish Functional Genomics Centre, University of Turku and Åbo Akademi and Biocenter Finland.

Lineage-depleted cells were cultured as described above for 3 or 24 h, and then O-propargyl-puromycin (OP-Puro; Click-iT Plus OPP AlexaFluor 488 kit, Life Technologies) was added (10 μM) to the medium for an additional 30 min. Cells were washed with PBS and stained with LIVE/DEAD Fixable Near IR Dead Cell Stain (ThermoFisher) as directed by the manufacturer. Cells were fixed in 0.5 ml of 1% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min covered on ice. Cells were washed in PBS, then permeabilized in 200 μl PBS supplemented with 3% FBS and 0.1% saponin (Sigma) for 5 min at room temperature. The azide-alkyne cycloaddition was performed using the kit as directed by the manufacturer. After the 30-min reaction, cells were washed in PBS supplemented with 3% FBS, then stained for surface markers indicated and analyzed by flow cytometry.

Bone marrow–derived neutrophils were plated in a 96-well plate at a density of 250,000 cells per well. The cells were then stimulated with HKCA (MOI = 1, 3, or 5), zymosan (5, 10, or 25 μg/mL), or left untreated for 6 hours at 37°C. After treatment, the supernatant was removed by centrifuging the plate at 500g for 5 minutes. To measure intracellular misfolded protein aggregates, cells were labeled with the cell-permeable fluorescent dye ThT (Abcam, catalog ab120751; stock 50 mM in DMSO, final concentration 5 μM in PBS). The cells were resuspended in 200 μL ThT staining solution and incubated for 15 minutes at room temperature in the dark. Cells were then washed twice with FACS buffer. Subsequently, the cells were Fc-γ receptor blocked using TruStain FcX (BioLegend, anti–mouse CD16/32, clone 93, catalog 101319) for 10 minutes at 4°C, and then stained for surface markers at 4°C in the dark for 30 minutes with the following antibodies: anti-CD45 (clone 30-F11 [RUO], BD Biosciences, catalog 562420), anti-CD11b (clone M1/70, BioLegend, catalog 101228), and anti-Ly6G (1A8 [RUO], BD Biosciences, catalog 741813). Cells were then washed with FACS buffer and stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific, catalog L34975) for live/dead discrimination. Flow cytometry was performed using a Fortessa-X20 instrument (BD Biosciences).

Lineage-depleted bone marrow cells were isolated for analysis of RNA content and cell size on day 0 or after 3 d of culture. At the time of analysis, cells were washed with PBS and stained in LIVE/DEAD Fixable Near IR Dead Cell Stain (ThermoFisher) as directed by the manufacturer, washed, and stained for surface markers indicated. For RNA content analysis by flow cytometry, cells were then washed, resuspended in 20 μg ml^-1 Hoechst 33342 (Cell Signaling Technologies) with 50 μg ml^-1 verapamil (Calbiochem) in PBS supplemented with 3% fetal bovine serum (FBS; GE Healthcare Life Sciences), vortexed briefly, and incubated covered at 37°C for 45 min. Pyronin Y (Sigma-Aldrich) was added directly to a final concentration of 1 μg ml^-1 and cells were briefly vortexed and incubated covered at 37°C for an additional 15 min. Cells were washed and immediately analyzed by flow cytometry.
For size measurement, HSCs were sorted and cultured as described above. Each day, cells were imaged on a Nikon Diaphot microscope equipped with a Nikon DS-Fi1 camera and NIS-Elements F imaging software (Nikon). Cell cross-sectional area was measured in ImageJ (NIH) calibrated to a micrometer, and volume was calculated assuming that cells are spherical.

Viability was determined using the LIVE/DEAD Fixable Near-IR Dead cell stain (ThermoFisher Scientific, Waltham, MA, USA). Epithelial purity was determined using human specific antibodies against CD45 (1:100; PerCP, HI30, Biolegend, San Diego, CA, USA) and EpCAM (1:500, Alexa 488, VU1D9, Cell Signaling Technology, Danvers, MA, USA). Cell surface markers were stained extracellular in 50µl FACS buffer (PBS supplemented with 2% FCS) for 15min on ice. Unspecific Fcγ receptor binding was blocked by human Fc block (CD16/CD32, BD Bioscience, Franklin Lakes, New Jersey, USA). Cells were analyzed using a FACS Canto II (BD Bioscience, Franklin Lakes, New Jersey, USA) and FlowJo software (TreeStar Inc.).