1.1 Single-cell suspensions from Mouse transplantable melanoma

DNase I （LS002139）
To prepare the 10 mg/mL stock solution, deoxyribonuclease I (DNase I) was dissolved in 100 mL of 5 mM calcium chloride(CaCl₂). Sterile fifilter the solution through a sterile 0.22 μm Stericup and store aliquots at −20°C.

 

Collagenase D （Roche,11088858001） 
The 40 mg/mL stock solution is prepared by dissolving 500 mg collagenase D in 12.5 mL Hank’s balanced salt solution (with Ca²⁺ and Mg²⁺). Sterile fifilter the solution through a sterile 0.22 μm membrane and store aliquots at −20°C.

 

10% BSA/PBS 
Dissolve 50 g of bovine serum albumin (BSA) in 500 mL PBS (can also be heated up to 40°C while stirring) and fifilter it through a 0.22 μm Stericup

 

Accutase (BD,561327)

 

100um细胞筛网（黄色）（Absin, abs7233)

1.对小鼠实施安乐死；

2.在开始解剖肿瘤之前，给小鼠注射70%的乙醇；

3.用剪刀通过矢状面切口切开小鼠，然后向后肢开侧向切口；

4.将皮肤与底层分离，显示皮下肿瘤；

5.用镊子切除肿瘤。

 

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1.将肿瘤转移到一个小培养皿(60×15mm)或一个6孔板中，填满5ml消化缓冲液(Hanks’ salt solution without Mg²⁺ and Ca²⁺ containing 2% FCS)，用剪刀和弯曲的镊子把肿瘤切成小块；

2.将肿瘤碎片悬液转移到50ml的试管中；用含5ml消化缓冲液清洗培养皿；(50ml tube containing 10ml digestion buffer with tumor tissue)；

3.将250的μg/ml胶原酶D和120的μg/ml DNase加入到10ml的组织消化缓冲液中；

4.在37°C的摇晃水浴中孵育45min；

5.加入500μl EDTA至最终浓度为10mM，停止消化。将细胞悬液过100μm筛网，用注射器的柱塞按压组织通过细胞过滤器；

6.用30ml的Würzburger缓冲液清洗细胞过滤器；

7.485×g离心5min，弃上清；

8.在2mlWürzburger缓冲液中重悬浮细胞；

9.计数。