70 μm nylon cell strainer

BM was flushed from the shafts of femurs and tibias from 6‐ to 8‐week‐old CD45.1⁺ C57BL/6 congenic mice, and single‐cell suspensions were prepared by passaging through a 70‐μm nylon cell strainer (Corning, Corning, NY, USA). Low‐density BM mononuclear cells (MNCs) were isolated by Ficoll‐Paque PLUS density gradient centrifugation (<1.077 g/ml; GE Healthcare, Uppsala, Sweden) to exclude granulocytes and red blood cells. Lineage‐positive cells expressing CD3, CD4, CD8 (T cells), CD11b (myeloid cells), B220 (B cells), Gr‐1 (macrophage/monocytes), CD71, TER119 (erythroid cells) or NK1.1 (natural killer cells) were removed from the MNCs by incubation with a rat anti‐mouse monoclonal antibody (mAb) cocktail as above (BD Biosciences, Franklin Lakes, NJ, USA), followed by incubation with sheep anti‐rat IgG‐conjugated magnetic beads (Dynal Inc., Oslo, Norway) with gentle agitation according to the manufacturer's protocol. Bead‐bound cells were removed using a magnetic particle concentrator (Dynal Inc.). The remaining lineage‐negative MNCs were considered as HSC‐eBMCs.

Mouse neuroblastoma N2a cells were grown in Dulbecco modified Eagle's medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 10%(V/V) fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C with 5% CO₂ in a humid incubator. Cerebral cortex neurons were prepared from neonatal rats within 72 h of birth. Cerebral cortex neurons were isolated as described previously (Zhu et al., 2016). Briefly, the cortices were digested with DMEM containing 2 mg mL⁻¹ papain (Invitrogen, Carlsbad, CA, USA) and 50 mg mL⁻¹ DNase (Sigma‐Aldrich, St. Louis, MO, USA) for 50 min at 37 °C. Digested tissues were filtered with a 70 μm nylon cell strainer (Corning, Corning, NY, USA). Isolated cells were seeded in 0.05 mg mL⁻¹ poly‐L‐lysine (Solarbio, Beijing, China)‐coated plates at a density of 2 × 10⁵ cells per well in a 24‐well plate. Neurons were cultured in DMEM/F12 (Gibco) containing 1% B27 serum‐free supplement (Invitrogen), and 1% penicillin/streptomycin (Gibco). After 48 h, 10 mm cytarabine (Sigma‐Aldrich) was added to suppress the growth of glial cells. Experimental treatments were performed after 7 days of culture.

Mice were culled 7–11 days after bacterial administration. Tumours were aseptically excised and halved. One half was placed in 10% buffered formalin and fixed for 24 h at RT. The other half was placed in 1 ml PBS (+ 0.05% l-cysteine for B. breve) and homogenised using a 70-μm nylon cell strainer (Corning). Cell strainers were washed with 1 ml PBS. Homogenised tumours were serially diluted with PBS and plated for retrospective counting as per [52 (link)].

The liver was briefly perfused with the buffer containing Hank’s buffered saline (HBSS) without Ca²⁺/Mg²⁺, 720 μM EDTA, 0.075% Na₂HCO₃), followed by Collagenase II solution (HBSS with Ca²⁺/Mg²⁺, 0.075% Na₂HCO₃, 1 mM CaCl₂, 1 mg/ml Collagenase Type II (Worthington, CLS-2)) at ∼1 ml/min for 5 min. After perfusion, livers were dissected and minced gently using forceps. Hepatocytes were washed with HBSS, filtered through 70 μm nylon cell strainer (Corning), and centrifuged at 50g for 3 min. Liver cell mixture was adjusted to 5 to 10 × 10⁶ cells/ml with complete L15 media (L15 pH 7.4, 0.72 mM HEPES, 60 mg/l glucose, and 5% FBS) and mixed with equal volume of Percoll solution (9:1 mixture of Percoll (GE, 17-0891-02) and 10XHBSS). The cell mixture was subject to centrifugation at 50g for 10 min at 4 °C. Hepatocytes were gently resuspended and washed with complete L15 media twice before plating on collagen-coated plates at the density of 1.2 × 10⁶/ml for culturing.

Spleen and liver samples (~2 mm²) were collected and cell suspensions were made by mechanical disruption and passage through a 70 μm nylon cell strainer (Falcon Corning, New York, USA) with 2 ml Dulbecco's Modified Eagle Medium supplemented with sodium pyruvate and glutamine (GE Healthcare, Glattbrugg, Zurich, Switzerland) and 10% Fetal bovine serum (FBS; Connectorate AG, Dietikon, Switzerland). Cell suspensions were transferred to a 50 ml conical tube containing glass beads and vortexed to crack open cells. 100 μl of cell suspension were transferred to a 24-well plate containing McCoy cells at confluence in DMEM supplemented medium, immediately centrifuged at 1790 RCF for 10 min and placed in an incubator (37°C, 5% CO₂) for 3 h. After incubation, 50 μg/ml of gentamycin (Bioconcept, Allschwil, Switzerland) and 30 μg/ml of ampicillin (ampicillin sodium salt, Sigma-Aldrich Chemie, Buchs, Switzerland) were added to the samples in order to inhibit growth of extracellular and other intracellular bacteria, respectively, as W. chondrophila is resistant to ampicillin at >32 μg/ml due to the presence of a β-lactamase (Goy and Greub, 2009 (link); Bertelli et al., 2010 (link)). Viability was assessed by qPCR and by light microscopy examination of McCoy cells 3 h or 7 days post-inoculation.