胰蛋白酶-EDTA (0.05%)，含酚红

通常情况下，胰酶可在冰箱中保存一周。不过，这一时间长度也随着这一周内的加热次数和使用频率而变化。同时，它也与细胞粘附的紧密程度有关，胰酶在非冰冻条件下每天都会丧失一定的活性。

TrypLE试剂不含动物源性与人源成份，纯度极高且对细胞的作用十分温和。该产品无需使用胰酶抑制剂对其进行终止。在含血清和不含血清的培养条件下，TrypLE试剂都是解离各类贴壁培养的哺乳动物细胞系的理想之选，能够直接做为胰酶替代品而应用于您当前的实验方案。

请通过此选择列表(https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin.html)来比较我们的细胞解离试剂。

是的，请访问此页面(https://tools.thermofisher.com/content/sfs/brochures/Phenol_RedFree_Media_Glance.pdf)浏览我们的无酚红培养基页面。

Dissociation of cells from Primary Tissue

TRYPSIN
1. After dissecting off unusable tissue, mince the remaining tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces by resuspending in a balanced salt solution without calcium and magnesium. Allow the tissue pieces to settle, and remove the supernatant. Repeat the wash 2 or 3 times.
2. Place the container with the tissue pieces on ice, and remove any remaining supernatant. Add 0.25% trypsin in a balanced salt solution without calcium or magnesium (1 ml of trypsin for every 100 mg of tissue).
3. Incubate at 4°C for 6 to 18 h to maximize penetration of the enzyme with little trypsin activity.
4. Decant and discard the trypsin from the tissue pieces. Incubate the tissue pieces with residual trypsin at 37°C for 20 to 30 min.
5. Add warm, complete media to the tissue pieces and gently disperse the tissue by pipetting. If using a serum-free medium, also add soybean trypsin inhibitor.
6. Filter the cell suspension through sterile, stainless steel mesh (100 to 200 µm) to completely disperse any remaining tissue. Count and seed the cells for culture.

COLLAGENASE
1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times with Hanks' Balanced Salt Solution (HBSS).
2. Add collagenase (50 to 200 U/ml in HBSS).
3. Incubate at 37°C for 4 to 18 h. Addition of 3 mM CaCl2 increases the efficiency of dissociation.
4. Filter the cell suspension through a sterile stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh collagenase can be added to the fragments if further disaggregation is required.
5. Wash suspension several times by centrifugation in HBSS.
6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

DISPASE
1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times in a calcium and magnesium-free balanced salt solution.
2. Add dispase (0.6 to 2.4 U/ml in calcium and magnesium-free balanced salt solution).
3. Incubate at 37°C for 20 min to several hours.
4. Filter the cell suspension through a sterile, stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh dispase can be added to the fragments if further disaggregation is required.
5. Wash suspension several times by centrifugation in the balanced salt solution.
6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

REFERENCE:
Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.