Anti cd11b microbead

Cells were prepared for ChIP-seq or ChIP-qPCR analysis according to Yamane et al. (2013) (link) with minor modifications. For each experiment, harvested cells were depleted of dead/apoptotic cells using the dead cell removal kit (Miltenyi Biotec) before further processing. For CEBPA experiments, myeloid cells were additionally enriched using anti-CD11B micro-beads (Miltenyi). Primary transformed and untransformed B cell precursors were typically harvested on day 7 after transduction; for CEBPA experiments, cells were harvested after 7 days of DOX addition. For library preparation, the NEBNext ChIP-Seq Library Prep Master Mix Set and NEBNext Multiplex Oligos for Illumina (NEB) were used. For RNA-seq, cells were isolated, cultured, and harvested as for ChIP-seq experiments. RNA was extracted in duplicate experiments from 5 × 10⁶ cells using the QIAGEN RNeasy mini kit. Libraries were generated from pre-enriched mRNA using the NEBNext mRNA Library PrepReagent Set for Illumina (NEB) and pre-amplified using the NEBNext Multiplex Oligos for Illumina (NEB). A detailed description of the experimental procedures, bioinformatic analyses, and statistics is available in the Supplemental Experimental Procedures.

Naïve CD4 T cells from 2D2 mice and CD11b⁺ cells from EAE-recovered mice were sorted using anti-CD4 microbeads and anti-CD11b microbeads, respectively (Miltenyi Biotec). The resulting CD4⁺ T cell-enriched population (1 × 10⁵ cells) was cocultured with the isolated CD11b⁺ cells (5 × 10⁴ cells) without MOG-peptide addition in a 96 well plate for 3 days. IL-2 levels in cell culture supernatants were determined using ELISA kits (eBioscience).

Subcutaneous, orthotopic or MMTV-PyMT tumours were excised, cut in small pieces, treated with 10 U ml⁻¹ collagenase I, 400 U ml⁻¹ collagenase IV and 30 U ml⁻¹ DNaseI (Worthington) for 30 min at 37 °C, squashed and filtered. Red blood cells were removed using erythrocyte lysis buffer and density gradients (Axis-Shield) were used to remove debris and dead cells.
Tumour-draining LNs were cut, dissociated with 10 U ml⁻¹ collagenase I, 400 U ml⁻¹ collagenase IV and 30 U mL⁻¹ DNaseI (Worthington) for 45 min at 37 °C and filtered.
Spleens were flushed with 200 U ml⁻¹ collagenase III (Worthington) and left for 30 min at 37 °C. Afterwards, spleens were filtered and red blood cells were removed using erythrocyte lysis buffer.
To purify DC subpopulations from tumour, spleen or LNs, CD11c⁺ cells were MACS-enriched (anti-CD11c microbeads; Miltenyi) and sorted using BD FACSAria II (BD Biosciences) according to the gating strategy in Fig. 1a, Supplementary Fig. 6A or Supplementary Fig. 8A, respectively.
Bone marrow leukocytes were isolated through flushing of tibia and femur. The obtained cell suspensions were filtered, and red blood cells were removed using erythrocyte lysis buffer. To purify bone marrow monocytes, CD11b⁺ cells were MACS-enriched (anti-CD11b microbeads; Miltenyi) before sorting.

To purify MDSCs, splenocytes from tumor‐bearing mice were stained with anti‐CD11b microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and enriched by positive selection using the MACS technique (Miltenyi Biotec). To obtain DO11.10 T cells, CD4⁺ T cells were isolated via negative selection using a CD4⁺ T‐cell isolation kit (Miltenyi Biotec).
For the isolation of tumor infiltrating leukocytes (TILs), solid tumors were isolated from CT26 tumor‐bearing mice. The tumors were fragmented and digested with collagenase D (Roche, Basel, Swiss) and DNase I (Roche) using the GentleMACS dissociator (Miltenyi Biotec), according to the manufacturer's recommendation. Subsequently, the cells were separated using 40%/70% Percoll (GE Healthcare Life Sciences, Chicago, IL, USA) gradient and leukocytes were obtained from the interphase.

CD11b⁺ cells were obtained by immunomagnetic cell sorting using anti-CD11b microbeads (Miltenyi Biotec), following manufacturer’s instruction, or by immunopanning. Single-cell RNA sequencing and data processing were performed as previously described [10 (link)].

The hippocampus was dissected and stored in 1 mL cold HBSS buffer (hippocampus from 5 mice for Sham and Surgery group, respectively). The hippocampus was minced and dissociated in 8 mL of a mixture containing collagenase IV (5 mg/ml), DNase I (5 mg/ml) and 5% FBS, followed by bathing in water at 37 °C for 30 min. Cell suspension was then filtered through 70 µm cell strainer (BD Falcon, USA). Myelin debris was removed by using Myelin Removal Beads II (Miltenyi Biotec, Germany). Cell suspension was centrifuged at 300 g/min for 10 min then resuspended in 0.5% BSA. After incubating with anti-CD11b microbeads (Miltenyi Biotec, Germany) on ice for 15 min, CD11b⁺ and CD11b⁻ cells were isolated by MiniMACS separator (Miltenyi Biotec, Germany) according to the manufacturer’s instructions.