Aria fusion

PBMC samples were stained with fluorescently labelled antibodies (Table S6) and sorted with BD ARIA Fusion with a 70-μm nozzle according to the gating strategy indicated (Fig S1).

Table S6. Antibodies.

Peripheral blood mononuclear cells were sorted into subsets at the University of Birmingham. The PBMCs were first thawed as described in the Supplementary Data, http://links.lww.com/HC9/A236. CD14⁺ monocytes were then selected using human CD14 MicroBeads (Miltenyi Biotec, Woking, UK) according to the manufacturer’s instructions; CD14⁺ cell pellets were frozen at −80^°C until RNA extraction. Non-CD14⁺ cells were resuspended in PBS and stained with live/dead marker (Zombie APC-Cy7 at 1:1000; Biolegend, San Diego, CA, USA) for 20 minutes at room temperature. Cells were washed and stained with the following fluorochrome-conjugated antibodies (BD Biosciences, UK): CD3-APC, CD4-PECF594, CD127-PECy7, CD25-BB515, CXCR3-PerCP-Cy5.5, CCR6-BV421, and CD19-PE for fluorescent-associated cell sorting (using BD Aria Fusion, BD, UK) of the immune cell subsets: (1) CD19⁺CD3^– B cells, (2) CD3⁺CD4⁺CD25^highCD127^low T_REG cells, (3) CD3⁺CD4⁺CCR6⁺CXCR3^– T_H17 cells, and (4) CD3⁺CD4⁺CCR6^–CXCR3⁺ T_H1 cells (Fig. S2, http://links.lww.com/HC9/A236). We used an anti-CD3 monoclonal antibody that did not induce T-cell activation (Fig. S3, http://links.lww.com/HC9/A236). All immune cell subsets were sorted into tubes containing RPMI−1640+10% FCS media kept at 4°C during the sorting period. After sorting, cells were pelleted and resuspended in RLT+βME buffer for lysis and RNA extraction.

MNC or CD34 enriched samples were centrifuged and resuspended in PBS/3%FBS containing an antibody panel consisting of: CD3/FITC, CD90/PE, CD49f/PECy5, CD38/PECy7, CD33/APC, CD19/A700, CD34/APCCy7, CD45RA/BV421 and Zombie/Aqua. Cells were stained (30 min at 4 °C) in the dark before washing and resuspension in PBS/3%FBS for cell sorting. For all samples, HSC/MPP pool cells (Lin⁻CD34⁺CD38⁻CD45RA⁻) were sorted using either a BD Aria III or BD Aria Fusion cell sorter (BD Biosciences) at the NIHR Cambridge BRC Cell Phenotyping hub. The gating strategy is illustrated in Extended Data Fig. 1b. The HSC/MPP population was treated as a single entity and not further subclassified in the analysis. The immunophenotypic HSC/MPP population includes both long-term, intermediate-term and short-term HSCs as well as multipotent progenitors, as demonstrated functionally in xenotransplantation assays^{51 (link)–54 (link)}.
In a subset of individuals, a small number of HPCs (Lin⁻CD34⁺CD38⁺) were also sorted. The antibody panel used is shown in Supplementary Table 2. The HPC cells were treated as a single entity in the analysis and not further subclassified. The immunophenotypic HPC compartment includes predominantly myeloid and erythroid progenitors.

Data for the following parameters were collected on the BD LSR II: forward scatter (FSC), side scatter (SSC) and SFGFP (33 (link)) fluorescence (488 nm excitation, 515–545 nm emission). Three to 10 ul of each sample was measured in high throughput mode. Each sample was required to have at least 5000 counts, but most had 10 000–50 000. Counts were gated in FSC versus SSC by choosing a window surrounding the largest cluster of cells. SFGFP fluorescence values were recorded in relative channel number (1–262,144 corresponding to 18-bit data) and the geometric mean over the gated data was calculated for each sample. Data for Figure 2C was collected on a BD Aria Fusion for the following parameters: forward scatter (FSC), side scatter (SSC), SFGFP fluorescence (488 nm excitation, 530 nm emission), and mRFP Fluorescence (561 nm excitation, 582 nm emission). SFGFP and mRFP fluorescence values were recorded in relative channel number (1–262,144 corresponding to 18-bit data) and the geometric mean over the gated data was calculated for each sample. Compensation was calculated automatically by the BD Aria FACSDiva software using the compensation setup feature.

GBM cells were labeled with phycoerythrin (PE)-conjugated monoclonal antibodies against human CD133 (CD133/2-PE, Milteny Bio) and FITC-conjugated monoclonal antibodies against human CD15 (CD15-FITC, Milteny Bio) at 4 °C for 10 min per manufacturer’s instructions, as we described previously [18 (link)]. Cells were then washed and resuspended in a stem cell growth medium consisting of Neurobasal media (Invitrogen), N2 and B27 supplements (0.5× each; Invitrogen), human recombinant bFGF and EGF (50 ng/mL each; R&D Systems) [3 (link)], penicillin G, and streptomycin sulfate (1:100; GIBCO-Invitrogen). CD133⁺/CD15⁺, CD133⁺, CD15⁺and CD133⁻/CD15⁻ cells were then flow-sorted with BD Aria Fusion. Dead cells were excluded by propidium iodide (PI) staining [18 (link)].

Gene expression analysis was carried out on liver macrophages using fluorescent activated cell sorting (FACS) followed by mRNA extraction (BD Aria Fusion). Briefly, cells were sorted into cold PBS, pelleted and resuspended in TRIzol for cell lysis followed by chloroform extraction and ethanol precipitation (Thermo Fisher Scientific, Mississauga, ON, Canada). Isolated mRNA was quantified using Nanodrop (Thermo Fisher Scientific, Mississauga, ON, Canada). Normalized concentrations of mRNA were then hybridized and sequenced using the preassembled Mouse Inflammation v2 gene profiling CodeSet and nCounter platform according to the manufacturers protocol (Nanostring Technologies, Seattle, WA, USA). Data was analyzed using nSolver software provided by Nanostring Technologies.