Gentlemacs tissue dissociator

Mice were killed and primary tumors (n=27), liver metastases (n=15) and PC (n=16) were retrieved in ice-cold PBS. Tumors were minced with scissors and dissociated enzymatically with the tumour dissociation kit mouse, (Miltenyi) and mechanically with the gentleMACS tissue dissociator (Miltenyi, Germany) according to the manufacturer’s instructions. The resulting single cell suspension was filtered through a 100 µm cell strainer (Greiner Bio-One) and erythrocytes were lysed with ACK lysis buffer (Gibco). Cells were counted and either used for organoid culture or FACS staining.
Human samples for FACS analysis were excised during routine pathological examination from patients with tumor surgery in 2021. Primary CRC samples (n=5), liver metastases (n=5) and PC (n=5) were cut in small pieces and enzymatically and mechanically dissociated with the tumor dissociation kit human (Miltenyi) and the gentleMACS tissue dissociator (Miltenyi) according to the manufacturer´s protocol. After dissociation, the resulting single cell suspension was filtered through a 70 µm cell strainer and erythrocytes were lysed with ACK lysis buffer (Gibco).

Single-cell suspensions for flow cytometry analysis were prepared as previously described [2 (link)]. Briefly, after anesthetization, blood was sampled from the eyes and leukocytes were isolated with Ficoll (Solarbio, R1017) according to the manufactures' instructions. Cells were filtered through a 40 μm cell strainer. To obtain single-cell suspensions from heart tissue, mice were perfused with saline through the LV to clear the remaining blood. Heart tissues were cut into 1 mm pieces and digested twice in the C-tube with collagenase II (1.5 mg/mL) and DNAse in gentleMACS™ Tissue Dissociators (Miltenyi Biotec). The heart tissue was centrifuged at 37°C for 30 min at a speed of 200 rpm. After removing debris, single-cell suspension was collected by mashing the digested tissue through a 70 μm strainer. For splenic and mediastinal lymph node (med-LN) cell preparation, spleens and med-LNs were isolated and mashed with the plunger of a 1 mL syringe through a 70 μm strainer. The collected cells were lysed with red blood cell lysis buffer (eBioscience, 00-4333-57) according to the instructions. Then, the cells were filtered through a 40 μm strainer, counted, and maintained on ice for further analysis.

Briefly, mice were anesthetized with 1% sodium pentobarbital (60 mg/kg, intraperitoneal injection), and the heart was exposed and perfused with pre-cold PBS on day 7 after MI. Then, the heart was cut into 1 mm and put into the C-tube with collagenase II solution (1.5 mg/mL, added 500 × DNAse). During broken twice using gentleMACS™ Tissue Dissociators (Miltenyi Biotec, USA), the heart tissue was centrifuged at 37 °C for 30 min. After removing debris, the supernatant was filtered (70 μm strainer) to obtain single cell suspension. Cells were stained with the following labeled antibodies: anti-mouse CD45 (Biolegend, Clone I3/2.3), CD19 (Biolegend, Clone 1D3/CD19), CD3 (Biolegend, Clone 17A2), CD4 (Biolegend, Clone GK1.5), F4/80 (Biolegend, Clone BM8), Ly-6G (Biolegend, Clone 1A8). Data were acquired on a FACS Beckman flow cytometer (BD Biosciences, USA), and analysis was performed with Flow Jo software (BD Biosciences, USA).