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.
