Apc ef780

Flow cytometry data was acquired on a FACSVerse using FACSuite software or FACSCanto, or LSR II Fortessa with FACS DIVA software (BD Biosciences). Data was analysed using Flowjo software version 9.9.6 (Treestar).
For cell surface staining antibodies conjugated to BV421, BV510, FITC, PE, PerCPCy5.5, PECy7, APC, and APCeF780 were obtained from BD Biosciences, eBioscience or Biolegend. Fc receptors were blocked using Fc block (BD Biosciences). Antibody clones were as follows: CD4 (RM4-5), CD8 (53–6.7), CD11b (M1/70), CD25 (7D4), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD69 (H1.2F), TCRbeta (H57-597), Thy1.2 (53–2.1), B220 (RA3-6B2), NK1.1 (PK136).
For Myc intracellular staining, cells were fixed and permeabilised overnight in PBS 1% FBS 0.5% PFA 0.2% Tween-20. Fix/perm was washed off and cells were stained with 1:200 rabbit anti-Myc antibody (Cell Signalling Technologies, clone D84C12, cat#5605S) for 1 hr at room temperature followed by 1:1000 anti-rabbit IgG (H+L) F(ab’)₂ AlexFluor647 secondary antibody (Cell Signalling Technologies, cat#4414S) for 1 hr at room temperature.
For IFNγ and Granzyme B intracellular staining cells were fixed and permeabilised using eBioscience Intracellular Fixation and Permeabilisation kit (eBioscience) as per manufacturer instructions. Cells were stained with anti-IFNγ (XMG1.2) and anti-Granzyme B (NGZB) at 1:100 and 1:200 respectively.

Flow cytometric analysis was performed as described previously (11 (link), 14 (link), 30 (link), 33 (link)). Briefly, following red cell-lysis (with 0.8% NH₄Cl buffer), BM, blood, spleen, MLN and adipose tissue cells were suspended in FACS buffer (PBS containing 2.5% BSA and 0.5 mM EDTA) and phenotyped using the following antibodies:
HSC analysis involved use of a dump channel (PE-conjugated lineage cocktail) in combination with FITC anti-Sca-1 and APC or biotin anti-CD117 antibodies. For surface marker staining, antibodies were used at 0.2 µg/10⁶ cells (1/100 dilution) except for anti-CD45 (1/200 dilution). Streptavidin was used at 1/500 dilution. Cell death was assessed by fixed viability stain (APC-ef780) or 7AAD (BD Bioscience, UK) staining. Data were acquired using FACS Canto or BD LSRII flow cytometers and analysed using FlowJo Software (Tree Star In, OR USA, version 8.8.7) and populations were gated using isotype and fluorescence minus one (FMO) controls (11 (link), 14 (link), 30 (link), 33 (link)). Exemplar gating strategies are shown in Supplementary Figure 1.

Flow cytometric analysis was performed as described previously (7 (link), 13 (link), 22 (link)). Briefly, following red cell-lysis (with 0.8% NH₄Cl buffer), bone marrow (BM) cells were suspended in FACS buffer (PBS containing 2.5% BSA and 0.5 mM EDTA) and phenotyped using the following antibodies/fluorophores, with gating as described previously (7 (link), 13 (link), 22 (link)) and shown in Supplementary Figure 1.
LSK HSC analysis involved use of a PE-conjugated lineage cocktail, in combination with FITC anti-Sca-1 and APC or biotin anti-CD117 antibodies. Antibodies were employed at 0.2 µg/10⁶ cells (1/100 dilution) except for anti-CD45 (1/200 dilution). Streptavidin was used at 1/500 dilution. Cell death was assessed by fixed viability stain (APC-ef780) or 7AAD (BD Bioscience, UK) staining. Data were acquired using a FACS Canto flow cytometer and analysed using FlowJo Software (Tree Star In, OR USA, version 8.8.7) with populations being gated using isotype and fluorescence minus one (FMO) controls (7 (link), 13 (link), 22 (link)).