Gentlemacs

Peripheral blood was collected from mice using the retroorbital bleeding route, and blood was collected into 4% sodium citrate (Sigma-Aldrich) to prevent clotting. RPMI + 10% FBS was added to dilute out the anti-coagulant, and then white blood cells were separated from red blood cells using centrifugation through histopaque-1083 (Sigma-Aldrich). The white blood cell layer at the interface between the histopaque and remaining medium was subsequently subjected to staining for flow cytometry analysis or sorting for scRNAseq.
Tumors were dissected and mechanically disaggregated. For flow cytometry validations, a GentleMACS (Miltenyi) was used for disaggregation, whereas for scRNAseq scissors were used to finely mince the tumors instead of the GentleMACS. The dissociated tissue was digested with collagenase type I (400 U/ml; Worthington Biochemical) for 20–30 min at 37°C. Samples were then passed through a 70-µm filter, and lymphocytes were enriched using centrifugation through a Percoll gradient (40% and 70%). The enriched lymphocyte layer at the 40%/70% interface was subsequently stained for flow cytometry or sorted for scRNAseq.

The lung tissue was dissociated using the gentleMACS in combination with the tissue dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Briefly, the lung was cut into the respective lobes and added into the dissociation tube with 2.4 mL of 1× buffer S, 100 μL of enzyme D, and 15 μL of enzyme A. Lung lobes were dissociated at 37°C for 30 min under rotation of the tube (program: 37C_m_LDK_1) in the gentleMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated cells were collected by centrifugation (400g, 10 min at 4°C) and red blood cells were lysed for 5 min at RT, washed and resuspended in culture media (eBioscience, ThermoFisher Scientific, California, USA). Finally, cells were filtered through a 70 μm MACS SmartStrainer (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell viability was assessed using DAPI staining after lung dissociation (S8C Fig).

For Mydgf recombinant protein experiments, CMs were isolated from hearts of WT mice at P1 using the Neonatal Heart Dissociation Kit in combination with gentleMACS^TM and Octo Dissociator (Miltenyi BioTec, Teterow, Germany) according to the manufacturer's instructions. CMs were plated into DMEM/F12 medium supplemented with 10% FBS at 37 °C and 5% CO₂. Then CMs were treated with Mydgf recombinant protein and harvested 16 hours later, and analyzed by immunofluorescence and RNA sequencing (RNA-Seq). For inhibition of Akt, CMs were treated with Akt inhibitor LY294002 (Sigma, USA). For siRNA experiments, CMs were respectively transfected with siRNA-Akt, siRNA-c-Myc, siRNA-FoxM1 or negative control (NC) using Lipofectamine 3000 (Invitrogen, Waltham, USA) transfection reagent as previously described 22 (link), harvested 48 hours later, and analyzed by western blot, immunofluorescence, or quantitative real-time Polymerase Chain Reaction (qRT-PCR). The siRNA-Akt sequences were: 5′-GCUACUUCCUCCUCAAGAATT-3′, 5′-UUCUUGAGGAGGAAGUAGCTT-3′, the siRNA-c-Myc sequences were: 5′-CCGUACAAGCCCUAUUUCAUTT-3′, 5′-AUGAAAUAGGGCUGUACGGTT-3′, the siRNA-FoxM1 sequences were: 5′-GCAAAUUUCCAGCCGGAAUTT-3′, 5′-AUUCCGGCUGGAAAUUUGCTT-3′, and the NC sequences were: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′.

We isolated the mouse hearts at 1 day after AR using the Neonatal Heart Dissociation Kit in combination with gentleMACS^TM and Octo Dissociator (Miltenyi BioTec, Teterow, Germany) according to the manufacturer's instructions. Then we used the Neonatal Cardiomyocyte Isolation Kit (130100825, Miltenyi BioTec, Teterow, Germany) to isolate mouse CMs by depletion of non-target cells. Nontarget cells were directly magnetically labeled with a cocktail of monoclonal antibodies conjugated with MACS MicroBeads. The magnetically labeled non-target cells were depleted by retaining them within a MACS (MS) column in the magnetic field of a MACS Separator, while the unlabeled CMs passed through the column. As for macrophages (MΦs) and endothelial cells (ECs), we incubated the target cells for 30 minutes at 4°C with the following antibodies: CD45-Percp (1:100, BD Biosciences, 557235), CD11b-PE (1:200, BD Biosciences, 557397), CD31-APC (1:200, Biolegend, 102419). After washing, we sorted the cells by flow cytometry on a FACS Aria Flow Cytometer (BD Biosciences, San Jose, CA, USA). We identified MΦs as CD45⁺CD11b⁺, ECs as CD31⁺.

Cardiac samples were collected at 3 or 8 days post-MI. Left ventricles (LV) were harvested after perfusion with physiologic serum at 37 °C. Infarcted Scar and Border Zones (BZ) were carefully separated and collected in 50 ml-falcon tube containing 5 ml of RPMI 1640 medium. Three LVs from same group were collected per tube. Digestion enzymes cocktail (5 mg of collagenase II (Gibco Cat#17101015), 6 mg of Dispase (Sigma-Aldrich Cat#D4693), 300 µg of DNAse I (Sigma-Aldrich Cat#DN25) per 5 ml) was added and sample were dissociated through GentleMACS^TM (Miltenyi biotec) for 15 min. After dissociation, samples were filtered through 70 µm and then 40 µm cell strainer and prepared for FACS staining in PBS-2% FBS buffer.