Collagenase 4

Tissue was surgically minced on a laboratory sterile table, and tissue fragments were preserved in MACS tissue storage until processing.
The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm³) on ice, and enzymatically digested with 500 U/ml collagenase I (SangonBiotech), 150 U/ml collagenase II (SangonBiotech), 50 U/ml collagenase IV (SangonBiotech), 0.1 mg/ml hyaluronidase (SangonBiotech), 30 U/ml DNaseI (SangonBiotech), and 5% Fetal Bovine Serum Origin South America (Yeasen) for 60 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300 g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).

Endometriotic tissues were collected in Department of Obstetrics and Gynecology in the First Affiliated Hospital of Xiamen University. The application of samples received permission from the ethics committee and all patients signed the informed consent. Endometriotic lesions were from a 35-year-old female suffered from ovarian endometriosis confirmed by laparoscopy and histopathology. This endometriosis patient had regular menstrual cycles and was without hormone treatment for more 3 months before the surgery.
The endometriotic samples were minced and digested by collagenase IV (#A004186–0001; Sangon Biotech; Shanghai, China) and deoxyribonuclease I (DNase I; #B002004–0005; Sangon Biotech; Shanghai, China). After being filtered through nylon cell-strainers with a 100 mesh which intercepted pieces of tissue, and then through a mesh size of 400 which blocked the epithelial glands cells and passed through the stromal cells. The epithelial and stromal cells were respectively cultured within DMEM/F12 medium containing 10% FBS in dishes at 10% CO₂ 37 °C incubation.

A single-cell suspension was prepared from the spleen or lymph node by passing the organ through a 70-μm nylon cell strainer, followed by treatment with red blood cell lysis buffer. Lung cell suspensions (right lobe) were prepared by perfusing cold saline containing heparin through the heart, removed, and sectioned in ice-cold medium. Dissected lung tissue was incubated in 0.7 mg/ml collagenase IV and 30 μg/ml DNase (Sangon Biotech (Shanghai), China) at 37°C for 30 min. Digested lungs were disrupted by passage through a 70-μm nylon cell strainer, treated with red blood cell lysis buffer, and processed over a 40:80% Percoll (GE Healthcare) gradient. The resulting cell suspension was washed and counted.

Lung cell suspensions were prepared by perfusing cold saline containing heparin through the heart, removed, and sectioned in ice-cold medium. Dissected lung tissue was incubated in 0.7 mg/mL collagenase IV and 30 µg/mL DNase [Sangon Biotech (Shanghai), China] at 37°C for 30 min. Digested lungs were disrupted by passage through a 70-µm nylon cell strainer, treated with red blood cell lysis buffer, and processed over a 40:80% Percoll (GE Healthcare) gradient. The resulting cell suspension was washed and counted.

Total MDSCs (T-MDSCs, CD11b⁺Gr-1⁺), M-MDSCs (CD11b⁺Ly6-C⁺) and PMN-MDSCs (CD11b⁺Ly6-G⁺) were isolated from the tumour tissues by the Myeloid-Derived Suppressor Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany), with all steps performed at 4 °C. Briefly, tumour-bearing mice fed the NSD or HSD for 16 days were sacrificed, and tumour tissues were harvested. Fresh tumour tissues were cut into pieces and then enzymatically digested with 0.2% collagenase IV (weight per volume) and 0.1% DNase (wt per vol) (Sangon Biotech, China) for 1 h at 37 °C. After the cells were filtered through 70 μM Nylon cell strainers (Falcon, USA), the resulting cell suspension was centrifuged at 93×g for 5 min. The cells were depleted of RBCs using RBC lysis buffer and washed twice with cold PBS containing 1% BSA. After treatment with FcR blocking reagent (50 μl per 10⁸ cells) for 10 min, cells were stained with the biotin-conjugated granulocyte receptor (GR)-1 or Ly-6G monoclonal antibody and further labelled with anti-biotin or streptavidin microbeads. Then, cells were passed through the MACS column (MS or LS separation column) for magnetic cell separation. The retained cells were M-MDSCs or PMN-MDSCs, and the purity and cell viability of the MDSC subfractions were typically greater than 90% (Supplementary Fig. 20a, b).

Spleens were minced through a 70 μm sieve and leukocytes were obtained after lysing red blood cells with ACK buffer. Livers were minced through a 100 μm cell strainer and digested for 30 min at 37 °C with rotation in HBSS (Ca²⁺, Mg²⁺) buffer containing 1 mg/ml (335 U/mg) collagenase IV (Sangon Biotech, Shanghai, China) and 200 μg/ml (100 Kunitz U/ml) DNase I (Sangon Biotech, Shanghai, China). Liver leukocytes were separated by percoll gradient centrifugation (GE Healthcare, Freiburg, Germany). The cells were then resuspended in RPMI-1640 at 1 × 10⁶/ml, and surface staining was performed as previously described [16 (link)].
F4/80 and CD11b were used to stain macrophages, CD11c and CD11b for DCs, and Ly6G and CD11b for granulocytes. CD11c-FITC (N418; Biolegend), Gr1-PE (RB6-8C5; Biolegend), CD11b-PerCP-Cy5.5 (M1/70; eBioscience), F4/80-APC (BM8; Biolegend), and CD3-Pacific Blue (145-2C11; eBioscience) antibodies were utilized. As for intracellular iNOS-PE (W16030C; Biolegend) staining, the fixation and permeabilization steps were followed by Intracellular Fixation & Permeabilization Kit (eBioscience). Flow cytometric acquisition was performed with a BD FACSVerse flow cytometer, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

The entire epididymis from the four mice was cleaned and washed three times with PBS and then divided into three parts: the caput, corpus, and cauda. The samples were stored in MACS Tissue Storage Solution for 48 h (Miltenyi, Bergisch Gladbach, Germany). Before dissociation, the epididymal tissues were cut into small pieces and transferred to 0.2% collagenase IV (1 mg/ml; Sangon, Shanghai, China) and DNase I (5 U/ml; Sangon, Shanghai, China) digestion solutions, followed by incubation at 37 °C for 15 min. After digestion and mechanical striking of single cells, the cell suspension was filtered through a cell strainer (Solarbio, Beijing, China) and converted into barcoded scRNA-seq libraries, according to the manufacturer’s protocol. The libraries were sequenced using the DNBSEQ-T7 (Mgi Tech, Shenzhen, China).