To date, there are no protocols optimized to the effective separation of spermatogonial stem cells (SSCs) from testicular cells derived from mouse testes, thus hindering studies based on mouse SSCs. In this study, we aimed to determine the most efficient purification method for the isolation of SSCs from mouse testes among previously described techniques. Isolation of SSCs from testicular cells derived from mouse testes was conducted using four different techniques: differential plating (DP), magnetic-activated cell sorting (MACS) post-DP, MACS, and positive and negative selection double MACS. DP was performed for 1, 2, 4, 8, or 16 h, and MACS was performed using EpCAM (MACSEpCAM), Thy1 (MACSThy1), or GFR α1 (MACSGFRα1) antibodies. The purification efficiency of each method was analyzed by measuring the percentage of cells that stained positively for alkaline phosphatase. DP for 8 h, MACSThy1 post-DP for 8 h, MACSGFRα1, positive selection double MACSGFRα1/EpCAM, and negative selection double MACSGFRα1/α-SMA were identified as the optimal protocols for isolation of SSCs from mouse testicular cells. Comparison of the purification efficiencies of the optimized isolation protocols showed that, numerically, the highest purification efficiency was obtained using MACSGFRα1. Overall, our results indicate that MACSGFRα1 is an appropriate purification technique for the isolation of SSCs from mouse testicular cells.

Abstract
 INTRODUCTION
 MATERIALS AND METHODS
  1. Animals
  2. Isolation of testicular cells from mouse testes
  3. Differential plating (DP) method
  4. Magnetic-activating cell sorting (MACS)
  5. Experimental design
  6. Alkaline phosphatase (AP) staining
  7. Statistical analysis
 RESULTS
  Effect of incubation time on the purification efficiency ofSSCs from mouse testicular cells using DP
  Effect of antibodies on the purification efficiency of SSCs frommouse testicular cells using MACS post-DP, MACS, positiveselection double MACS, and negative selection MACS
  Determination of an optimized technique for the isolationof SSCs from mouse testicular cells
 DICUSSION
 REFERENCES

• Na Rae Han(Department of Animal Life Science, Kangwon National University)
• Hye Jin Park(Department of Animal Life Science, Kangwon National University)
• Hyun Lee(Department of Animal Life Science, Kangwon National University)
• Jung Im Yun(Institute of Animal Resources, Kangwon National University)
• Kimyung Choi(Optipharm Incorporation)
• Eunsong Lee(College of Veterinary Medicine, Kangwon National University)
• Seung Tae Lee(Department of Animal Life Science, Kangwon National University, Department of Applied Animal Science, Kangwon National University) Correspondence