9036-06-0

Experimental Procedure for Isolating Primary Hematopoietic Stem Cells (HSCs) from Mice Using Pronase E^[2]1. Animal Anesthesia and Abdominal Exposure: (1) Anesthetize male or female mice. (2) Under sterile conditions, expose the liver via abdominal incision. 2. Liver Perfusion Procedure: (1) Perform liver perfusion through the portal vein using Hanks' Balanced Salt Solution (HBSS) to remove blood and impurities. (2) Conduct enzymatic perfusion with Pronase E and collagenase to dissociate liver tissue. 3. Liver Extraction and Cell Release: (1) Carefully extract the liver from the abdominal cavity using sterile forceps, and quickly transfer it to a sterile culture dish. (2) Gently peel the liver capsule and apply mild mechanical agitation to release cells from the liver tissue. 4. Cell Digestion and Preliminary Centrifugation: (1) Suspend the released cells in a mixed enzyme solution containing Pronase E, collagenase, and DNase, and digest at 37°C for 20 minutes. (2) Perform two rounds of low-speed centrifugation (50 g, 3 minutes) to separate liver cells, and collect the cell suspension. 5. HSC Separation and Density Gradient Centrifugation: (1) Further centrifuge the cell suspension (450 g, 8 minutes) and collect the pellet. (2) Resuspend the pellet in 18% Nycodenz solution as the basis for density gradient separation. (3) Construct a density gradient with 12% Nycodenz, 8.2% Nycodenz, and Gey's Balanced Salt Solution (GBSS). (4) Isolate and purify HSCs from the 8.2% Nycodenz layer through density gradient centrifugation. Note: This protocol provides standard operating procedures; please adjust and optimize according to specific experimental needs and conditions.