组织培养用酶 Dispase 是切断肽链的中性及非极性氨基酸 N 端的金属蛋白酶，能够把上皮细胞从组织上剥离成片状，长期以来用于原代培养的细胞分离与分散，近年来也被应用于再生医疗领域。与胰蛋白酶和胶原酶等蛋白酶的作用不同，Dispase 造成的细胞损伤更少，可温和地分散细胞，因此在 ES 和 iPS 细胞等新兴领域中被应用于细胞的分散和传代。
产品信息
| 项目号 | D1516119
| D1516122
|
|---|
| 品名 | 分散酶Ⅰ
| 分散酶Ⅱ
| | 包装 | 结晶酶，无菌产品
| 粉末酶，非无菌产品
| | 酶活性 | 10,000–13,000 PU/vial
| 300-360 PU/mg enzyme powder
| | 添加剂等 | 含有醋酸钙 | 含有糊精和醋酸钙 | | 储存方法及有效期 | 生产后在 2～8℃下可储存 2 年（未开封）；开封溶解后请冻存在 - 20℃以下，并在 6 个月内使用。 | 生产后在 2～8℃下可储存 2 年（未开封）；开封溶解后请冻存在 - 20℃以下，并在 6 个月内使用 |
|
主要特点
- Dispase 是 Paenibacillus sp. (旧名：Bacillus polymyxa) 来源的中性金属蛋白酶，活性中心含有 Zn²⁺，并使用 Ca²⁺稳定其活性。
- 酶活性几乎不受血清影响，无论有无血清均可使用。添加 EDTA 等螯合剂、使用新鲜培养基或缓冲液清洗细胞即可停止酶促反应 。
- 可以很好地分解组成基底膜的 Ⅳ 型胶原、纤连蛋白，使上皮细胞从组织上剥离成片状。
- 与胰蛋白酶、胶原酶等蛋白酶的作用不同，细胞损伤少，可温和地分散细胞。
- 作用条件范围广泛，可以在不同的浓度、处理时间、温度以及 pH 条件下起作用。
- 在一般细胞培养温度 (37℃) 下非常稳定，对于部分类型细胞，添加本产品至培养基可实现悬浮培养 。
- 本产品无动物源成分，不含支原体。
使用方法
分散酶Ⅰ ：需在无菌条件下溶解制备。打开铝盖，用消毒乙醇对瓶塞及其周围区域进行消毒，使用已灭菌的注射器吸取所需量的培养基或缓冲液，注入瓶中进行溶解。铝盖容易划伤手指，操作时请注意安全。
分散酶Ⅱ：称量所需量的分散酶Ⅱ粉末，使用培养基或缓冲液溶解。在洁净环境下进行 0.2 μm 过滤灭菌。
作用条件
| 酶浓度 | 100 ~ 2000 PU/mL | | 溶解缓冲液 | 含有 Ca²⁺的缓冲液或者培养基（可含血清）
| | 反应时间 | 30 min ~ 几天 | | 反应温度 | 室温～37℃ | | 反应 pH | 6.5 ~ 9.0 | | 抑制剂 | EDTA、Fe³+、Fe²+、Ni²+、Cu²+、Al³+、Zn²+
* 本酶的活性中心含有 Zn²⁺，如反应溶液中含有过量的 Zn²⁺，则酶活性可能受到抑制 |
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活性检测法
将 1 mL 酶溶液 (50 PU/mL，含 2 mM 醋酸钙的 50 mM Tris-HCl 缓冲液) 添加到 5 mL 0.6% 酪蛋白水溶液 (pH 7.5，含 2 mM 醋酸钙的 50 mM Tris-HCl 缓冲液) 中，在 30℃下反应 10 min。反应结束后，加入 5 mL 三氯乙酸试剂终止反应。在 30℃下静置 30 min 并进行过滤后，检测 275 nm 吸光度。
在此条件下，1 PU (Protease Unit) 定义为 1 min 内水解酪蛋白产生相当于 1 μg 酪氨酸的酶量。(活性单位 (PU) 指 Protease Unit)
注）三氯乙酸试剂：使用蒸馏水溶解无水醋酸钠 1.8%(w/v)、三氯乙酸 1.8%(w/v)、醋酸 1.98%(w/v) 后，将 pH 调整到 4.0 后得到的试剂。
使用注意事项
1. 本产品为研究用试剂，请勿将本产品用于人或动物的活体实验以及诊断用途。
2. 本产品为分散酶，接触到眼睛、口腔、皮肤或粘膜等部位可能引起炎症。如不慎直接接触，请立即用大量流水进行冲洗，必要时尽快就医。
3. 本产品为保证酶稳定性，成分中含有醋酸钙。因此本产品在高浓度的磷酸缓冲液中使用时会产生白色沉淀 (钙盐)，酶活性和酶稳定性可能下降。
4. 对于酶溶液和作用条件，推荐使用含有胎牛血清的培养基，有利于分散以及分散后的细胞增殖。
5. 打开铝盖时容易划伤手指，操作时请注意安全。
6. 请将本产品置于规定的储存条件下进行储存，请勿使用过期产品。
7. 请将 Dispase 溶液于 - 20℃下冷冻储存，并在 6 个月内使用完毕。
8. 废弃本产品时，请遵守废弃物处理的相关条例，妥善处理。
Q&A
1. 关于 Dispase Ⅰ 和 Dispase Ⅱ
Q1： Dispase Ⅰ 和 Dispase Ⅱ的区别？
A1：
Dispase Ⅰ：结晶酶，无菌，含醋酸钙；
Dispase Ⅱ：粉末状酶，非无菌，含糊精和醋酸钙（溶解后根据需求进行过滤灭菌）。
※ Ⅰ和Ⅱ的纯化程度略有不同（Ⅰ的纯化程度更高）。
Q2：Dispase Ⅰ 和 Dispase Ⅱ 在使用时有哪些注意事项？是否适用于不同的实验体系？
A2：可用于多种细胞类型和实验体系，目前尚未有不适用的实验体系情况反馈。需注意，因包装不同，开封后应根据存储条件决定如何使用产品。
Dispase Ⅰ：真空密封，基本上需一次性使用完毕；
Dispase Ⅱ：粉末分装使用。
2. 关于使用方法
Q1：推荐浓度是多少？
A1：推荐浓度如产品说明书所示：100～2,000 PU/mL。建议可先在该浓度范围内摸索合适的浓度。
Q2：将上皮细胞剥离至薄片时的推荐浓度为？
A2：酶的效果取决于细胞类型和培养条件，对此无法提供明确的推荐浓度。请参考产品说明书中的浓度100-2000 PU/mL，摸索合适的使用浓度。此外，浓度相同时， DispaseⅠ 和 Dispase Ⅱ在使用上没有明显差异。
Q3：酶活性、稳定性与Ca²⁺相关，那在不含Ca²⁺的PBS（-）或 HBSS中是否还能发挥作用？
A3：如产品说明书所述，在磷酸盐缓冲溶液中使用时，可能出现白色沉淀或酶活性、稳定性会降低。因此，需尽量避免使用磷酸盐缓冲溶液，建议在含有Ca²⁺条件下使用该产品。
Q4：可以使用PBS (-)吗？
A4：可以使用，但酶活性和稳定性可能会降低。请调整酶添加量（酶浓度）并在溶解后立即使用。
Q5：使用时Ca²⁺的推荐浓度是多少？
A5：仅需几mM即可充分发挥作用。
Q6：前面提到使用含钙的缓冲液或培养基作为酶的溶解液，如果使用含胎牛血清（FBS）的培养基，浓度是否可以调整为10%？
A6：可以。
3. 关于作用
Q1：除了可分解IV 型胶原蛋白和纤连蛋白外，是否还可分解其他的基底膜蛋白？
A1：对层粘连蛋白有轻微的分解作用。
Q2：经过 Dispase 散酶处理后是否会对细胞存活率或细胞基因造成影响？
A2：细胞存活率因条件不同会有较大差异。 而对基因的影响尚未得到证实。
Dispase, a protease for tissue culture, is a metalloproteinase that cleaves the N-terminus of neutral and non-polar amino acids in peptide chains. It can detach epithelial cells from tissues in sheet form and has long been used for the isolation and dispersion of cells in primary culture. In recent years, it has also been applied in the field of regenerative medicine. Unlike proteases such as trypsin and collagenase, Dispase causes less cellular damage and can disperse cells gently, thus being used for cell dispersion and subculture in emerging fields such as ES and iPS cell research.
Product Information
| Product Code | D1516119 | D1516122 | | Product Name | Dispase I | Dispase II | | Form | Crystallized enzyme, sterile product | Powdered enzyme, non-sterile product | | Enzyme Activity | 10,000–13,000 PU/vial
| 300-360 PU/mg enzyme powder
| | Additives | Contains calcium acetate | Contains dextrin and calcium acetate | | Storage & Shelf Life | Store at 2–8°C for 2 years (unopened). After reconstitution, store frozen at ≤ -20°C and use within 6 months. | Store at 2–8°C for 2 years (unopened). After reconstitution, store frozen at ≤ -20°C and use within 6 months. |
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Key Features
1. Dispase is a neutral metalloproteinase derived from Paenibacillus sp. (formerly Bacillus polymyxa), with Zn²⁺ in its active center and Ca²⁺ for stabilizing its activity.
2. Enzyme activity is hardly affected by serum and can be used in the presence or absence of serum. The enzymatic reaction can be terminated by adding chelating agents such as EDTA or washing cells with fresh medium or buffer.
3. It can effectively decompose type Ⅳ collagen and fibronectin that constitute the basement membrane, detaching epithelial cells from tissues in sheet form.
4. Unlike trypsin, collagenase and other proteases, it causes less cellular damage and enables gentle cell dispersion.
5. It has a wide range of working conditions and can function under different concentrations, treatment times, temperatures and pH values.
6. It is highly stable at the general cell culture temperature (37°C). For some cell types, suspension culture can be achieved by adding this product to the medium.
7. This product is animal-origin free and mycoplasma-free.
Usage Method
Dispase I: Dissolve and prepare under sterile conditions. Remove the aluminum cap, disinfect the rubber stopper and its surrounding area with sterile ethanol, draw the required volume of medium or buffer with a sterilized syringe, and inject it into the vial for dissolution. Note safety during operation as the aluminum cap may cause finger scratches.
Dispase II: Weigh the required amount of Dispase II powder and dissolve it with medium or buffer. Perform 0.2 μm filtration sterilization in a clean environment.
Working Conditions
Enzyme Concentration
| 100 ~ 2000 PU/mL | Dissolution Buffer
| Ca²⁺-containing buffer or medium (serum may be included)
| Reaction Time
| 30 minutes ~ several days
| Reaction Temperature
| Room temperature ~ 37°C
| Reaction pH
| 6.5 ~ 9.0 | Inhibitors
| EDTA、Fe³+、Fe²+、Ni²+、Cu²+、Al³+、Zn²+
* The active center of this enzyme contains Zn²⁺; excessive Zn²⁺ in the reaction solution may inhibit the enzyme activity |
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Activity Assay Method
Add 1 mL of enzyme solution (50 PU/mL, 50 mM Tris-HCl buffer containing 2 mM calcium acetate) to 5 mL of 0.6% casein aqueous solution (pH 7.5, 50 mM Tris-HCl buffer containing 2 mM calcium acetate), and react at 30°C for 10 minutes. After the reaction, add 5 mL of trichloroacetic acid reagent to terminate the reaction. Let it stand at 30°C for 30 minutes, filter it, and detect the absorbance at 275 nm.
Under these conditions, 1 PU (Protease Unit) is defined as the amount of enzyme that hydrolyzes casein to produce 1 μg of tyrosine per minute.
Note: Trichloroacetic acid reagent is prepared by dissolving 1.8% (w/v) anhydrous sodium acetate, 1.8% (w/v) trichloroacetic acid and 1.98% (w/v) acetic acid in distilled water, and adjusting the pH to 4.0.
Precautions for Use
- This product is a research reagent and shall not be used for in vivo experiments on humans or animals, nor for diagnostic purposes.
- This product is a dispase and may cause inflammation if it comes into contact with eyes, mouth, skin or mucous membranes. In case of accidental direct contact, rinse immediately with a large amount of running water and seek medical attention as soon as necessary if needed.
- Calcium acetate is contained in this product to ensure enzyme stability. Therefore, white precipitation (calcium salt) may form when this product is used in a high-concentration phosphate buffer, which may lead to a decrease in enzyme activity and stability.
- For the enzyme solution and working conditions, it is recommended to use medium containing fetal bovine serum, which is conducive to cell dispersion and proliferation after dispersion.
- Note safety during operation as the aluminum cap may cause finger scratches when opened.
- Store this product under the specified storage conditions and do not use expired products.
- Store the Dispase solution at -20°C in a frozen state and use it up within 6 months.
- When discarding this product, comply with relevant waste disposal regulations and handle it properly.
Q&A
1. About Dispase Ⅰ and Dispase Ⅱ
Q1: What is the difference between Dispase Ⅰ and Dispase Ⅱ?
A1:
- Dispase Ⅰ: Crystallized enzyme, sterile, contains calcium acetate.
- Dispase Ⅱ: Powdered enzyme, non-sterile, contains dextrin and calcium acetate (perform filter sterilization after dissolution as required).
Note: The purification degrees of Ⅰ and Ⅱ are slightly different (Dispase Ⅰ has a higher purification degree).
Q2: What precautions should be taken when using Dispase Ⅰ and Dispase Ⅱ? Are they suitable for different experimental systems?
A2: They can be used for a variety of cell types and experimental systems, and no feedback of incompatible experimental systems has been received to date. Note that due to different packaging forms, the usage of the product after opening should be determined in accordance with the storage conditions.
- Dispase Ⅰ: Vacuum-sealed, basically needs to be used up in one single operation.
- Dispase Ⅱ: The powder can be aliquoted for use.
2. About Usage Method
Q1: What is the recommended concentration?
A1: The recommended concentration is as indicated in the product manual: 100～2,000 PU/mL. It is recommended to optimize the appropriate concentration within this range first.
Q2: What is the recommended concentration for detaching epithelial cells into sheets?
A2: The enzymatic effect depends on cell types and culture conditions, so a definitive recommended concentration cannot be provided. Please refer to the concentration range of 100–2000 PU/mL in the product manual to optimize the appropriate working concentration. In addition, when used at the same concentration, there is no significant difference between Dispase Ⅰ and Dispase Ⅱ in application.
Q3: Since the enzyme activity and stability are related to Ca²⁺, can it still exert its effect in Ca²⁺-free PBS (-) or HBSS?
A3: As stated in the product manual, white precipitation may occur or the enzyme activity and stability may decrease when used in phosphate buffer solutions. Therefore, the use of phosphate buffer solutions should be avoided as much as possible, and it is recommended to use this product under Ca²⁺-containing conditions.
Q4: Can PBS (-) be used?A4: It can be used, but the enzyme activity and stability may decrease. Please adjust the enzyme dosage (enzyme concentration) and use it immediately after dissolution.
Q5: What is the recommended concentration of Ca²⁺ for use?
A5: Only a few mM is sufficient for it to exert its effect fully.
Q6: It was mentioned earlier that calcium-containing buffer or medium is used as the enzyme dissolution solution. If a medium containing fetal bovine serum (FBS) is used, can its concentration be adjusted to 10%?
A6: Yes.
3. About Function
Q1: In addition to decomposing type Ⅳ collagen and fibronectin, can it decompose other basement membrane proteins?
A1: It has a slight decomposing effect on laminin.
Q2: Will treatment with Dispase affect cell viability or cellular genes?
A2: Cell viability varies greatly under different conditions. The effect on genes has not been confirmed yet.
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