Collagenase 2 and 4

Fresh pituitary adenoma tissues were minced with the Iris scissor into small pieces and digested for 30 min at 37 °C, 800 rpm, with a digestion solution containing PBS and collagenase II and IV (1.5 mg/ml, Gibco). The cell suspension was further filtered through 45-µm nylon mesh to remove cell aggregates and re-suspended in L15 with 10% FBS. Then, a second enzymatic digestion with accutase to dissociate the remaining cell clusters into single cells was performed. Finally, add L15 medium enriched with 10% fetal bovine serum (FBS).

At 24 hours post-CLP procedures, 10⁷ MenSCs were labeled with PKH26 (Sigma-Aldrich, St. Louis, MO, USA) in accordance with the protocol of the manufacturer. Labeling efficiency was 95 % as validated by flow cytometry. Labelled MenSCs (2 × 10⁶ cells/mouse) were resuspended in 250 μl of PBS and injected intraperitoneally. Animals were euthanized at 24 hours post-injection, and different organs and fluids, including the spleen, heart, kidneys, lungs, liver, and intraperitoneal fluid, were recovered and incubated at 37 °C for 30 minutes with 250 U/ml of collagenase II and IV (Gibco). The single-cell suspensions obtained were resuspended into 400 μl of cytometry buffer (PBS 1×, bovine serum albumin (BSA) 0.2 %, sodium azide 0.01 %) and then analyzed by flow cytometry.