70 μm mesh strainer

Mice that underwent SNTM were euthanized at 7 and 14 days after repair to assess the early and middle phases of the immune responses to PNI. The regenerative bridge and its distal ScN segment (~10 mm length) were cut and placed in a petri dish with 1 mL of RPMI-1640 (Corning) and then cut into 1 mm pieces. Then, the tissues were transferred to a 50-mL conical tube with 10 mL of digestion buffer (comprised of 3 mg/mL collagenase type I [Sigma, C1-22], 1 mg/mL hyaluronidase [Sigma, 37326-33-3], and 0.5 mL of 1 mM HEPES in RPMI-1640). The digestion was performed for 1 h in a 37 °C shaker, and we obtained a single-cell suspension via a 70-μm mesh strainer (BD Biosciences). RBC blood cell lysis buffer (00-4333-57) was used for RBC lysis, and cells were incubated with membrane antibodies (anti-CD45, anti-CD11b, anti-F4/80). Subsequently, washed cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4 °C. Once fixed, cells were incubated with intracellular antibodies. The excluded antibody markers were Ly6G (granulocytes), while the positive selection markers for macrophages included CD45 (total lymphocytes), CD11b (bone marrow-derived monocytes), F4/80 (macrophages), iNOS (M1), and CD206 (M2). The gating strategy was shown in Supplemental Fig. 2.

C57/BL6J were euthanized 5 days after immediate or delayed CP-TIB repair with a 5 mm conduit sciatic repair. The regenerative bridge was harvested within the conduit and digested for 1 h in 37 °C water bath in digestion buffer. The single-cell suspension was obtained by straining through a 70-μm mesh strainer (BD Biosciences). The cells were centrifuged at 300 × g for 10 min and then resuspended in 0.5% BSA (Sigma) in DPBS. The number of cells in each sample was counted using hemocytometer. The cells were then incubated for 1 h in 37 °C water bath with bioparticles (pHrodo™ Green Zymosan bioparticles, 75 particles per cell). The number of particles per cell was selected based on pilot titration study. After the incubation, the cells were washed two times with DPBS and were plated on a v-bottom 96-well plate (Nunc, Thermo Scientific) for antibody staining and FACS, as described^{34 (link),38 (link)}. Briefly, macrophages were identified using the four antibodies (F4/80, CD14, CD16/32, CD11b), and the phagocytosis performance of the macrophages was evaluated using the GFP signal related to phagocytosed bioparticles. The outcome measures were assessed using t test. Significance was set as P < 0.05 throughout.

Human peripheral blood was collected to isolate PBMC using Ficoll‐Hypaque (MP Biomedicals, Santa Ana CA, USA) gradient centrifugation. Human GCs and FF were obtained from patients with bPOI and controls undergoing IVF/ICSI‐ET.
Murine lymphoid tissues (spleen and draining lymph nodes) were thoroughly minced and consecutively passed through the 70‐μm mesh strainer (BD Biosciences, San Jose, CA, USA) to obtain single‐cell suspensions. To prepare single cell suspension from ovary, ovaries from two mice were isolated, mixed and cut into small pieces, followed by enzymatic digestion for 20 minutes at 37°C in plain RPMI buffer (HyClone, Thermo Fisher Scientific, Waltham, MA, USA) with Collagenase IV (4 mg/ml; Gibco, Thermo Fisher Scientific) and DNase (0.01 mg/ml; Sigma, Louis, MO, USA), and then mashed through 70‐μm cell strainers.
Immature female C57BL/6 mice (3‐week‐old) were injected with 200 IU pregnant mare serum gonadotropin (PMSG, SANSHENG, Ningbo, Zhejiang, China) by intraperitoneal to stimulate follicle growth for 44 h. The ovaries were removed and primary GCs were isolated and harvested from large antral follicles by needle puncture.