Streptavidin particles plus

Bone marrow cells from mice were labeled with a cocktail of biotin-conjugated lineage marker antibodies in combination with Streptavidin Particles Plus (BD, 557812) according to the manufacturer’s instructions. Lineage-positive cells were depleted using Cell Separation Magnet (BD, 552311) according to the manufacturer’s instructions to obtain lineage-negative (Lin−) bone marrow cells. To arrest cells at metaphase, ~2.5 × 10⁶ Lin− cells were incubated in complete RPMI media containing 100 ng/ml nocodazole (Sigma) at 37 °C for 2 h. Cells were then resuspended in 5 ml of 0.056 M KCl and incubated for 30 min at room temperature. KCl-treated cells were then resuspended in 1 ml of cold methanol: glacial acetic acid (3:1) solution and spun down at 1000 rpm for 10 min at 4 °C for fixation. The fixation step was repeated for three times, and every time 200 μl of solution was left when removing the supernatant. After the last fixation step, the metaphase cell suspension can be stored at −20 °C for at least 1 year. To prepare metaphase chromosome spreads, 20 μl of metaphase cell suspension was released onto a pre-chilled glass slide with the help of a pipette from 1.5-m height. The spreads were air dried and used for H3K27me3 IF staining and X-paint DNA FISH.

Fetal liver cells were incubated with biotin-conjugated anti-TER119 (BD Biosciences) and biotin-conjugated anti-CD45 (BD Biosciences) antibodies on ice for 30 min. After wash, cells were reacted with Streptavidin Particles Plus (BD Biosciences) on ice for 30 min. The reacted sample was added into the 2 mL IMag buffer (PBS containing 0.5% BSA and 2 mM EDTA), and TER119⁺/CD45⁺ hematopoietic cells were removed by a Cell Separation Magnet (BD Biosciences).

A mixed lymphocyte reaction was performed as previously described^{48 (link)}. BMDMs were seeded into 24-well plates. After transfection with si-β-catenin or siR, macrophages were stimulated with PBS (M0), LPS + INF-γ (M1) or IL-4 (M2) for 24 h. Then, cells were irradiated with 20 Gy X-rays. Naïve T cells were negatively sorted from the lymph nodes of allogeneic mice using biotin-labeled B cell- and myeloid cell-specific antibodies, followed by magnetic Streptavidin Particles Plus (BD IMag^TM, Franklin Lake, New Jersey, USA). T cells were labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE, Invitrogen) and added into irradiated differentially polarized BMDMs at a ratio of 5:1.5 days later, nonadherent cells were harvested and stained with APC anti-CD8 antibody (BD Pharmingen). Proliferation of CD8⁺ T cells was analyzed by FACS.

BM was harvested from primary transplanted NSG mice at 24 weeks after transplant. BM underwent magnetic mouse CD45 antibody depletion using Biotin Rat anti–mouse CD45 (BD Biosciences, 553078) and Streptavidin Particles Plus (BD Biosciences, 557812) and was transplanted 1:1 i.v. into irradiated (280 cGy) NSG mice. Secondary mice were analyzed at 12 weeks after transplant.

Mouse monocytes were first enriched from blood or bone marrow mononuclear cells by negative selection using biotinylated anti-Ly6G, B220, CD3, NK1.1, and Ter119 antibodies (Supplementary Table 5) and magnetic beads (Streptavidin particles plus, BD Biosciences). Monocyte subsets were then sorted using Aria III, Aria-Fusion or Influx (BD Biosciences and BD Diva Software) cell sorters using Streptavidin-PE, CD11b-Pacific blue, CD115-BV605, and Ly6C-APC antibodies (Supplementary Table 5) as Lin⁻ (CD3, B220, Ter119, Nk1.1, and Ly6G-biotin/Streptavidin-PE⁺ cells) CD11b⁺, CD115⁺ monocytes, and separated according to Ly6C expression (Supplementary Figure 3B). Human CD14⁺ cells were sorted from mononuclear cells using CD14 microbeads and the AutoMacs system (Miltenyi Biotech). Monocytes subsets were first enriched from mononuclear cells by negative selection using CD19⁻, CD56⁻, CD3⁻, CD16⁻ microbeads and the AutoMacs system (Miltenyi Biotech), and then sorted using a cell sorter and CD45, CD14, and CD16 antibodies after exclusion of CD56⁺ cells (Supplementary Table 5). CD34⁺ cells were sorted from human cord blood samples using magnetic beads and the AutoMacs system (Miltenyi Biotech).