In rodents, fibroblast-like PLCs appear in the testis by postnatal day 14–21, while ALCs in testis are formed on postnatal days 49–56. Therefore, PLCs were isolated from 3-week-old male KM mice and ALCs were isolated from 8-week-old male KM mice.57 (link) Briefly, decapsulated testes were digested with collagenase type IV (Biosharp, China) at 1 mg/mL for 7 min at 37°C shaking water bath. The isolated cells were cultured in DMEM (Gibco, USA) with 10% FBS (ExCell Bio, China) for 1 h, and then the complete medium was changed to remove blood cells, spermatocytes, and Sertoli cells that adhered slowly. Subsequently, the adherent cells were cultured in a cell incubator at 37°C in 5% CO2. After 24 h, the cells were treated with hypotonic solution (20 mM Tris, pH 7.4) for 2 min to remove myoid cells. Cells were continually cultured for another 48 h to obtain LCs for subsequent experiments.
Collagenase type 4
Manufactured by Biosharp
Sourced in China
Collagenase type IV is an enzyme used to digest the collagen present in the extracellular matrix of tissues. It is commonly utilized in cell isolation and tissue dissociation procedures.
Automatically generated - may contain errors
2 protocols using collagenase type 4
1
Isolation of Testicular Leydig Cells
2
Subcutaneous Tumor Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell isolation was performed according to a previous procedure60 (link)–62 (link) with some adaptations. Briefly, after 4 mice were sacrificed, subcutaneous tumors were collected and cut into small pieces (<2 mm in diameter) and then digested with 10 ml of dissociation solution (RPMI 1640 medium containing 10% FBS, 1 mg/mL collagenase type IV (Biosharp, Hefei, China), 100 µg/mL DNase I (Biosharp, Hefei, China), and 2.5 µg/mL hyaluronidase (Biosharp, Hefei, China)) for 60 min on a 37 °C shaker. Next, 4 ml RPMI 1640 medium containing 10% FBS was added to dilute the suspensions. Then, the cell suspensions were passed through 70 µm cell strainers, and the lower layer was collected after centrifuging at 400 × g for 10 min. Subsequently, the erythrocytes were lysed with red blood cell lysis buffer (Biosharp, Hefei, China) according to the manufacturer’s instructions and then washed and resuspended the remaining single cells in PBS.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!