Clone d7

For HPC isolation, lineage-negative cells were sorted from the BM of C57BL/6 mice followed by staining with Sca-1 (FITC, Biolegend, clone D7, dilution 1:100, lot 108106) and c-kit (PE, Biolegend, clone 2B8, dilution 1:100, lot 105808) antibodies. To differentiate HPCs into MDSCs, 2 × 10⁵ Lin^-Sca-1^-C-kit⁺ HPCs were placed in each well of 24-well plates and cultured in SFEM (STEMCELL Technologies) medium containing GM-CSF (10 ng/mL; PeproTech) and IL-6 (10 ng/ml; PeproTech). At different time points during culturing, 5 μM GSK343 (Selleck Chemicals) was added to the culture system and the newly generated CD11b⁺Gr-1⁺ cells were analyzed at 96 h.

In order to determine cell numbers in the bone marrow, around 100.000 bone marrow cells were freshly isolated. Aliquots of bone marrows were washed once in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄), resuspended in 100 μL H/S, Fc-receptors were blocked by incubation for 30 min with Fc-block (anti-mouse CD16/CD32, Biolegend #101302), samples were washed once and aliquots were stained with FITC-coupled anti-mouse CD45 antibodies (1:200, clone RA3-6B2; Biolegend, #103228) and APC-coupled antibodies against mouse CD4 (1:200, clone GK1.5; Biolegend, #17-0041-81) or APC-coupled antibodies against mouse CD8 (1:200, clone 53-6.7; Biolegend, #17-0081-81) or PE-coupled antibodies against mouse CD19 (1:200, clone GD5; Biolegend, #115508) or APC-coupled antibodies against mouse Ly6G (Gr1) (1:200, clone RB6-BL5; Biolegend, #103107). In addition, cells were stained with Alexa 647-coupled anti-mouse CD45 antibodies (1:200, clone 30-F11; Biolegend, #103228) and FITC-coupled antibodies against mouse Sca-1 (1:200, clone D7; Biolegend, #108105). Cells were stained for 1 h at 4°C, washed once in H/S and analyzed on a FACS-Calibur. We analyzed the expression of the above described markers on 100 000 cells by flow cytometry.

Flow cytometry experiments were performed using a BD FACS Fortessa. Populations of mature blood cells were identified by staining PB and BM for B220 (BioLegend, clone RA3-6B2), CD3 (Thermo Scientific, clone 145-2C11), Gr1 (BioLegend, clone RB6-8C5) and Mac1 (BioLegend, clone M1/70). Analysis of erythroid precursors in PB and BM was conducted with antibodies against Ter-119 (BioLegend, clone TER-119) and CD71 (BioLegend, clone R17217). Stem and progenitor cells in BM were identified as previously described [5 –7 ] by staining with a cocktail against lineage markers (BioLegend, B220, CD3, Gr1, Mac1 and Ter119) as well as for c-Kit (eBioscience, clone 2B8), Sca1 (BioLegend, clone D7), CD34 (BioLegend, clone MEC14.7), Fc-γ-II/III-R (eBioscience, clone 93), Thy1.1 (BioLegend, clone OX7) and Flt3 (eBioscience, clone A2F10). Gating strategies were determined by fluorescence minus one staining [8 ].