Collagenase type 4

We used differential centrifugation to extract exosomes from tissues and leucorrhea. Briefly: ectopic tissue was disaggregated into a single cell suspension with type IV collagenase (Solarbio, China). Leucorrhea was diluted with PBS to make a mixed solution. The supernatant and leucorrhea solution were centrifuged at 4 °C with a high-speed centrifuge (Thermo, USA) at 500 g for 10 min to remove living cells, 2000 g for 10 min to remove dead cells, and 10,000 g for 20 min to eliminate the cell debris. Every step was repeated twice. The supernatant was then centrifuged at 100,000 g twice with ultracentrifuge (Beckman, USA) for 70 min each time. The exosomes were resuspended or lysed with different reagents for subsequent experiments.

Brain tissues were placed into 500 ug/ml collagenase type IV (Solarbio, China; dissolved in RPMI 1640 medium containing 10% FBS) and cut into several pieces with a surgical scalpel. After digestion at 37°C for 1 h, cells were pressed through a 70‐μm cell strainer. Myelin debris was removed, and single‐cell suspension was acquired by gradient density centrifugation (30%/70% Percoll solution; GE Healthcare, USA). For surface staining, the brain cells were incubated with CD45‐APC‐Cy7 (Biolegend, USA) and CD4‐FITC (Biolegend, USA) for 30 min. For intracellular staining, cells were fixed and permeabilized with Fixation Buffer (Biolegend, USA) and Intracellular Staining Perm Wash Buffer (Biolegend, USA) according to the manufacturer's guidelines, followed by GZMB‐APC/Per‐Cy7 (Biolegend, USA) staining for 40 min. The eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (00‐5523‐00; Thermo Scientific) and Foxp3‐PE antibody (Biolegend, USA) were utilized for intranuclear staining in Treg cells. Flow cytometry analysis was performed by CytoFLEX cytometer (Beckman, CA). The data were analyzed with CytExpert and FlowJo software.

Cecum samples were first freed from residual fat tissue, Peyer’s patches, feces and then cut into smaller pieces and incubated in Hanks’ Balanced Salt Solution with 2% FCS, 5 mM of EDTA and 2 mM of dithiothreitol for 30 min at 37°C and vortexed. The inter-epithelial lymphocytes (IEL) fraction was dissected by filtering over a 70 μm cell strainer. To recover the lamina propria lymphocytes (LPL) fraction, IEL-depleted intestine pieces were washed in Hanks’ Balanced Salt Solution supplemented with 2% FCS and enzymatically digested for 45 min at 37°C with Collagenase type IV (Solarbio), Neutral protease and DNase I (Solarbio) in 1640 medium. Single-cell suspensions were generated by filtering over a 70-μm cell strainer. The IELs and LPLs were purified by density centrifugation on a 67% and 44% percoll gradient (Cytiva).

GMSCs collection adhered to the Declaration of Helsinki and associated regulations. Consent forms were signed by all volunteers, with ethical approval granted by the Ethics Committee of the Stomatological Hospital of Southern Medical University (Approval No.: EC-CT-[2022]41).
The isolation and culture of GMSCs followed a modified protocol from Zhang et al. (Zhang et al., 2009 (link)). Gingival tissues, acquired from healthy individuals aged 23–30 years, were carefully segmented into 1–2 mm³ pieces and thoroughly rinsed with sterile PBS. Subsequently, the tissues were incubated with Dispase II (Solarbio, Beijing, China) overnight at 4 °C to separate the epithelial layer from the underlying connective tissue. Collagenase Type IV (Solarbio) digestion was then carried out at 37 °C for 1 h. The centrifuged pellet was then resuspended in Dulbecco’s Modified Eagle Medium supplemented (DMEM; Gibco, MA, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P/S; Gibco). The cells were cultured in T25 flasks at 37 °C with 5% CO₂. Passages were performed using 0.25% trypsin when the cell confluence reached 70%–80%. Experiments were conducted using cells at passages 3-6.