胰蛋白酶 (Trypsin)是由胰脏产生没有活性的胰蛋白酶原分泌到小肠后，小肠内的肠肽酶会活化该酶原，形成胰蛋白酶，经常用于动物组织的培养细胞消化或者一些组织的消化，但对细胞有的潜在损害，尤其是在37℃环境下危害较大。
无酶细胞消化液 (含酚红)不含胰蛋白酶等蛋白消化酶类，但能有效地使贴壁细胞与培养瓶/皿表面脱离而达到分离细胞目的。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
其特点是：
1、作用温和；
2、对细胞的损伤和破坏极小，不影响细胞生物学特性，是肿瘤细胞的极好细胞脱壁方法；
3、可以在血清存在的情况下进行消化。消化后的细胞可进行传代培养，亦可用于提取核蛋白和胞浆蛋白、Western Blot、免疫共沉淀等实验，通常室温下10min左右就可以消化下大多数贴壁细胞，该消化液适用于消化肿瘤、脑、肝、肾、肺组织等，尤其适用于上皮组织。
自备材料：
1、PBS、培养液
2、显微镜、离心机
操作步骤 (仅供参考)：
1、贴壁细胞的消化
①吸除培养液，用无菌PBS、培养液洗涤细胞1次。
②加入少量无酶细胞消化液 (含酚红)，略盖过细胞即可 (一般按细胞的有效体积的10倍添加)。
③室温放置10min，如置于37℃脱壁反应会加速，直到细胞完全脱壁，不同的细胞消化时间有所不同；亦可显微镜下观察，细胞明显收缩，并且肉眼观察培养器皿底部发现细胞的形态发生明显的变化或者用枪吹打细胞发现细胞刚好可以被吹打下来，吸除消化液。
④加入细胞培养液或5倍体积PBS缓冲液终止反应。如果发现消化不足，则加入无酶细胞消化液 (含酚红)重新消化。
⑤1000-2000g离心3-5min，沉淀细胞，弃上清，尽量去除无酶细胞消化液 (含酚红)，加入含血清的完全培养液重新悬浮细胞，即可用于后续实验。
2、组织的消化
①PBS、培养液清洗组织1次，用无菌的刮勺或茶匙将剪碎的组织碎片转移至合适容器。
②按组织有效体积的5-10倍，加入无酶细胞消化液 (含酚红)。
③置于37℃作用4-48h，无需振荡，不同的细胞消化时间有所不同；对于较难分解的肿瘤细胞，可作用5天或更长时间，但应重新用消化液悬浮。
④吹打组织碎片数次，释放松散的细胞，轻轻晃动培养瓶或皿，倒置显微镜下检查分离情况，当细胞量少和/或在组织碎片中仍可见细胞时，需要继续消化。
⑤1000-2000g离心3-5min，沉淀细胞，弃上清，尽量去除无酶细胞消化液 (含酚红)，加入含血清的完全培养液重新悬浮细胞，即可用于后续实验。
注意事项：
1、尽量减少反复冻融的次数，以免失效。
2、试剂开封后请尽快使用，以防影响后续实验效果。
3、在使用细胞消化液的过程中，要特别注意避免消化液被细菌污染。
4、细胞消化液消化细胞时间不宜过长，否则细胞铺板后生长状况会较差。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Trypsin is secreted as inactive trypsinogen by the pancreas into the small intestine, where it is activated by enteropeptidase to form active trypsin. It is commonly used for dissociating cultured cells from animal tissues or digesting certain tissues, but it poses potential damage to cells, with more severe harm particularly at 37℃.
Enzyme-Free Cell Dissociation Solution (Phenol Red Included) contains no proteolytic enzymes such as trypsin, yet it can effectively detach adherent cells from the surface of culture flasks/dishes to achieve cell separation. This reagent is for research use only (RUO) and is not intended for clinical diagnosis or any other purposes.
Features:
1. Gentle action on cells.
2. Minimal damage and disruption to cells, without affecting cellular biological properties, making it an excellent method for detaching tumor cells.
3. Enables cell dissociation in the presence of serum. Dissociated cells can be subjected to subculture, or used for experiments including nuclear and cytoplasmic protein extraction, Western Blot, and co-immunoprecipitation. Most adherent cells can be detached within approximately 10 minutes at room temperature. This solution is suitable for digesting tumor, brain, liver, kidney, and lung tissues, and is especially applicable to epithelial tissues.
Materials to Be Prepared by Users
1. PBS buffer, cell culture medium
2. Microscope, centrifuge
Operating Procedures (For Reference Only)
1. Dissociation of Adherent Cells
① Aspirate the culture medium and rinse the cells once with sterile PBS buffer or culture medium.
② Add a small amount of Enzyme-Free Cell Dissociation Solution (Phenol Red Included), just enough to cover the cells (generally, add 10 times the effective volume of the cells).
③ Incubate at room temperature for 10 minutes; the detachment reaction can be accelerated at 37℃ until the cells are completely detached, with the incubation time varying for different cell types. Alternatively, observe the cells under a microscope: remove the dissociation solution once the cells show obvious shrinkage, visible morphological changes at the bottom of the culture vessel to the naked eye, or can be readily detached by pipetting.
④ Add cell culture medium or 5 volumes of PBS buffer to terminate the reaction. If dissociation is insufficient, add fresh Enzyme-Free Cell Dissociation Solution (Phenol Red Included) and repeat the process.
⑤ Centrifuge at 1000-2000×g for 3-5 minutes to pellet the cells. Discard the supernatant, remove residual Enzyme-Free Cell Dissociation Solution (Phenol Red Included) as thoroughly as possible, and resuspend the cells in complete culture medium containing serum for subsequent experiments.
2. Digestion of Tissues
① Rinse the tissues once with PBS buffer or culture medium, and transfer the minced tissue fragments to an appropriate container using a sterile spatula or spoon.
② Add Enzyme-Free Cell Dissociation Solution (Phenol Red Included) at 5-10 times the effective volume of the tissues.
③ Incubate at 37℃ for 4-48 hours without shaking; the incubation time varies for different tissues. For hard-to-digest tumor cells, the incubation time can be extended to 5 days or longer, with resuspension in fresh dissociation solution required during this period.
④ Pipette the tissue fragments several times to release the loosened cells. Gently shake the culture flask or dish and check the dissociation status under an inverted microscope. If the number of released cells is small and/or cells are still visible within the tissue fragments, continue the digestion process.
⑤ Centrifuge at 1000-2000×g for 3-5 minutes to pellet the cells. Discard the supernatant, remove residual Enzyme-Free Cell Dissociation Solution (Phenol Red Included) as thoroughly as possible, and resuspend the cells in complete culture medium containing serum for subsequent experiments.
Precautions
1. Minimize repeated freeze-thaw cycles to prevent reagent inactivation.
2. Use the reagent as soon as possible after opening to avoid compromising subsequent experimental results.
3. Exercise extreme caution to prevent bacterial contamination of the cell dissociation solution during use.
4. Avoid prolonged incubation of cells with the dissociation solution, as this may lead to poor cell growth after plating.
5. For your safety and health, wear a lab coat and disposable gloves during operation.
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