Live dead viability dye

Transcriptional profiling of MDDCs was performed using whole genome RNA sequencing. MDDCs from four healthy blood donors were infected or stimulated as indicated in the figure legends, stained with anti-human CD86 (BD Biosciences) and a live/dead viability dye (ThermoFischer) before sorting into serum on a FACSAria II (BD Biosciences). Cells were lysed in TRIzol reagent (Thermo Fisher), RNA was isolated according to the manufacturer's instructions, and the samples were submitted to HudsonAlpha Institute for Biotechnology (https://hudsonalpha.org/) for library preparation and RNA-sequencing. Fifty bp-length single-end sequences were aligned to the human genome (hg38) using STAR version 2.4.2a. Samtools 0.1.19 was used to filter alignments to a MAPQ score threshold of 30. Counts per gene were called using feature Counts version 1.4.6 and gencode v24 genome annotation. Samtools 0.1.19 was used to filter alignments to a MAPQ score threshold of 30.

For flow cytometric analysis, cells were first incubated with Fc receptor block (BD Biosciences, for human cells) or anti-CD16/32 (eBioscience, for mouse cells) with a LIVE/DEAD viability dye (Thermo Fisher Scientific) for 10 min at 4 °C in FACS buffer (PBS/0.5% BSA/2 mM EDTA), followed by staining with antibody panels for 30 min at 4 °C. The following human antibodies with corresponding isotype controls were used: the PD-L1 monoclonal antibody (Cat. No. 329705) was purchased from BioLegend. The results of flow cytometry were analyzed using FlowJo software.

Flow cytometry analysis was used to assess cell surface markers listed in  "Reagents and Antibodies" section as well as determine the number of PHK26 and PKH67 positive cells in the lungs, liver, and spleen during adoptive transfer. For the analysis of cell surface markers, harvested cells were first incubated with FcR-Blocking solution (eBioscience) for 15 min on ice. Next, samples of 2 × 10⁶ cells were incubated with appropriate antibody panels for 30 min on ice. Cells were then washed and analyzed using the Fortessa X-20 (Becton Dickson).
To detect labeled macrophages in tissue, the lungs, liver, and spleen were digested using 2mg/mL collagenase II (Sigma-Aldrich, St Louis, MO, USA) as described above in "Adoptive Transfer of Monocytes in the Model of Endotoxemia" section. Cell suspension was next pre-cleaned via filtering through a 70μm cell strainer. Cells were incubated with live/dead viability dye for 30 min on ice (Thermo Fisher, Waltham, MA, USA). PKH26 and PKH67 labeled macrophages within the digested organs were analyzed with flow cytometry (Fortessa X-20) and imaging flow cytometry (Image Stream Mark II, Amnis). For analysis of αM on labeled macrophages, preparation was carried out as above.
Imaging flow cytometry analysis results were analyzed using IDEAS 6.2 software. The PKH26 and PKH67 labeled cells were captured on channels 2 and 3, respectively.