本产品包含各种酶法组织消化中常用的胶原酶等蛋白酶,能特异性地降解胞外胶原蛋白,使胶原蛋白和纤维粘连 蛋白的网状结构解离,而不损坏细胞表面结构的完整性。使用本产品进行组织消化可以使组织中细胞间的桥连结构松动,使组织由块状变成絮状,经过机械吹打后制成少量细胞群团和大量单个细胞的细胞悬液。制备的细胞悬液可用于原代培养、流式细胞仪进行细胞生物学 (如凋亡、增殖、细胞周期等)研究。
适用范围:各种正常和肿瘤组织,从中分离得到原代细胞,包括上皮细胞,间质细胞,成纤维细胞等。
使用说明
1. 提前将消化酶从-20℃冰箱取出置于2-8℃融化;
2. 使用消化酶稀释液 (货号D1521149)按照1: 1比例稀释组织消化酶,配制成工作浓度;
3. 将组织转移至培养皿中,加入3 mL组织清洗液 (货号O1521152)冲洗组织,反复2-3次;
4. 在无菌条件下将组织机械切割成1~2 mm³ 大小之后转移至15 mL无菌离心管中;
5. 加入5 mL组织清洗液,1500 rpm常温离心3 min;
6. 弃上清,加入12 mL工作浓度的消化酶,标记样本名称及编号并封口,放入恒温振荡培养箱中消化,设置参数37℃、200~300 rpm (根据样本质地设置转速),待组织消化呈团絮状,没有明显肉眼可见硬块终止消化;
7. 取出消化好的样本,1500 rpm常温离心3 min。
8. 弃上清后,加入6 mL含血清培养基进行终止消化,1500 rpm常温离心3 min。
9. 弃上清后加入稀释液待用。
注意事项
1. 本产品在使用前需在2-8℃溶化后使用,融化后需在一周内使用完毕。
2. 使用产品时应注意无菌操作,避免污染。
3. 本产品储存浓度为工作浓度的2倍,使用前需要用稀释液按照1: 1稀释。
4.为保持本产品的最佳使用效果,一般在37℃条件下使用,根据具体实验情况也可在2-8℃条件下使用。
5. 请于保质期内使用本产品,超出保质期后,将无法达到理想的使用效果。
6. 本产品仅用于科研用途,不能用于体外诊断。
This product contains proteases such as collagenase commonly used in enzymatic tissue digestion. It can specifically degrade extracellular collagen, dissociate the network structure of collagen and fibronectin without damaging the integrity of cell surface structures. Tissue digestion with this product loosens intercellular bridging structures, transforming massive tissues into flocculent substances. After mechanical pipetting, cell suspensions containing small cell clusters and a large number of single cells can be obtained. The prepared cell suspensions are applicable to primary cell culture and cytological researches by flow cytometry, including apoptosis, proliferation, cell cycle and other assays.
Application Scope
It is applicable to various normal and tumor tissues for isolation of primary cells, including epithelial cells, stromal cells, fibroblasts and other cell types.
Instructions for Use
1. Take the digestive enzyme out of the -20℃ refrigerator in advance and thaw it at 2–8℃.
2. Dilute the tissue digestive enzyme at a ratio of 1:1 with digestive enzyme diluent (Catalog No. D152114) to prepare working concentration solution.
3. Transfer tissues into a petri dish, add 3 mL tissue washing solution (Catalog No. O1521152) to rinse tissues, and repeat the washing process 2–3 times.
4. Under aseptic conditions, mechanically cut tissues into 1–2 mm³ fragments, then transfer them into sterile 15 mL centrifuge tubes.
5. Add 5 mL tissue washing solution, and centrifuge at 1500 rpm for 3 minutes at room temperature.
6. Discard the supernatant, add 12 mL working-concentration digestive enzyme. Label sample names and numbers, seal the tubes, and place them in a constant-temperature shaking incubator for digestion. Set parameters at 37℃, 200–300 rpm (rotation speed adjusted according to tissue texture). Terminate digestion when tissues become flocculent with no visible hard lumps.
7.Take out digested samples and centrifuge at 1500 rpm for 3 minutes at room temperature.
8. Discard the supernatant, add 6 mL serum-containing medium to terminate digestion, and centrifuge at 1500 rpm for 3 minutes at room temperature.
9. Discard the supernatant and add diluent for subsequent use.
Precautions
1. The product shall be thawed at 2–8℃ before use and consumed within one week after thawing.
2. Strict aseptic operation is required during use to avoid contamination.
3. The storage concentration of this product is twice the working concentration, and it must be diluted 1:1 with special diluent before application.
4. To maintain optimal performance, the product is generally used at 37℃. It can also be applied at 2–8℃ depending on specific experimental requirements.
5. Use the product within the shelf life. Expected experimental effects cannot be guaranteed beyond the expiration date.
6. This product is for scientific research only and shall not be used for in vitro diagnosis.