ST-1是MTT的一种升级替代产品，和MTT或其它MTT类似产品如XTT、MTS等相比有明显的优点。首先，MTT被线粒体内的一些脱氢酶还原生成的formazan不是水溶性的，需要有特定的溶解液来溶解；而WST-1和XTT、MTS产生的formazan都是水溶性的，可以省去后续的溶解步骤。其次，WST-1产生的formazan比XTT和MTS产生的formazan更易溶解。再次，WST-1比XTT和MTS更加稳定，使实验结果更加稳定。另外，WST-1和MTT、XTT等相比，线性范围更宽，灵敏度更高。
本试剂盒可以用于细胞因子等诱导的细胞增殖检测，也可以用于抗癌药物等对细胞有毒试剂诱导的细胞毒性检测，或一些药物诱导的细胞生长抑制检测等。本试剂盒检测非常便捷。无须使用同位素，所有的检测步骤仅在同一块96孔板内完成。不必洗涤细胞，不必收集细胞，也不必采用额外步骤去溶解formazan。可以用于大批量样品的检测。酚红和血清对本试剂盒的测定无明显影响。
WST-1对细胞无明显毒性。加入WST-1显色后，可以在不同时间反复用酶标仪读板，使检测时间更加灵活，便于找到最佳测定时间。
产品组份
W1507451 | 组分 | 100T | 500T | 2500T | 储存条件 | W1507451A | WST-1 (粉末) | 1瓶 | 1瓶 | 1瓶 | -20℃, 避光保存。 | W1507451B | 电子耦合试剂 | 1mL | 5 mL | 25 mL | -20℃, 避光保存。 |
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使用方法
1. WST-1溶液的配制：把电子耦合试剂加入到WST-1粉末中，完全溶解即成WST-1溶液。WST-1溶液4ºC避光可保存一周，而不影响使用效果。短期内不使用的WST-1溶液，分装后可以-20ºC避光保存半年(尽量避免反复冻融)。冻存的WST-1溶液溶解后可能会观察到一些沉淀物，这是正常现象，37ºC水浴孵育2-10分钟，通常可以完全溶解。
2. 通常细胞增殖实验每孔加入100微升2000个细胞，细胞毒性实验每孔加入100微升5000个细胞(具体每孔所用的细胞的数目，需根据细胞的大小，细胞增殖速度的快慢等因素决定)。按照实验需要，进行培养并给予0-10微升特定的药物刺激。
3. 每孔加入10微升WST-1溶液。如果起始的培养体积为200微升，则需加入20微升WST-1溶液，其它情况以此类推。可以用加了相应量细胞培养液和WST-1溶液但没有加入细胞的孔作为空白对照。
4. 在细胞培养箱内继续孵育0.5-4小时，对于大多数情况，孵育1-2小时就可以了。时间长短根据细胞的类型和细胞的密度等实验情况而定，初次实验时可以在0.5、1、2和4小时后分别用酶标仪检测，然后选取吸光度范围比较适宜的一个时间点用于后续实验。
5. 把96孔板置于摇床上摇动一分钟，以充分混匀待检测体系。
6. 在450nm测定吸光度。如无450nm滤光片，可以使用420-480nm的滤光片。可以使用大于600nm的波长作为参考波长进行双波长测定。
注意事项：
1. 由于使用96孔板进行检测，如果细胞培养时间较长，一定要注意蒸发问题。一方面，由于96孔板周围一圈最容易蒸发，可以采取弃用周围一圈的办法，改加相同量的PBS、水或培养液；另一方面，可以把96孔板置于靠近培养箱内水源的地方，以缓解蒸发。
2. 本试剂盒的检测依赖于脱氢酶催化的反应，所以还原剂(例如一些抗氧化剂)会干扰检测，如果待检测体系中存在较多的还原剂，需设法去除。
3. 用酶标仪检测前需确保每个孔内没有气泡，否则会干扰测定。
4. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
WST‑1 is an upgraded alternative to MTT. It has significant advantages over MTT and other MTT‑related products such as XTT and MTS.First, the formazan produced by the reduction of MTT by some dehydrogenases in mitochondria is not water‑soluble and requires a special solubilization solution.In contrast, the formazan produced by WST‑1, XTT, and MTS is water‑soluble, eliminating the subsequent solubilization step.Second, the formazan generated by WST‑1 is more soluble than that produced by XTT and MTS.Third, WST‑1 is more stable than XTT and MTS, leading to more stable experimental results.In addition, compared with MTT and XTT, WST‑1 has a wider linear range and higher sensitivity.
This kit can be used for cell proliferation assays induced by cytokines,as well as cytotoxicity assays induced by cytotoxic agents such as anticancer drugs,or cell growth inhibition assays induced by various drugs.The assay is highly convenient.No radioisotopes are required, and all detection steps can be performed in a single 96‑well plate.
There is no need to wash or harvest cells, or to perform additional steps to dissolve formazan.
The kit is suitable for high‑throughput detection.Phenol red and serum have no significant effect on the assay.
Components
W1507451 | Components | 100T | 500T | 2500T | Storage | W1507451A | WST-1(powder) | 1 vial | 1 vial | 1 vial | -20℃, Store in the dark. | W1507451B | Electron Coupling Reagent | 1 mL | 5 mL | 25 mL | -20℃, Store in the dark. |
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Instructions
1. Preparation of WST‑1 solution:
Add 1 mL of electron coupling reagent to the WST‑1 powder and dissolve completely to obtain the WST‑1 solution.The WST‑1 solution can be stored at 4°C protected from light for one week without affecting performance.For long‑term storage, aliquot the solution and store at ‑20°C protected from light for up to six months (avoid repeated freeze‑thaw cycles).Some precipitation may be observed after thawing, which is normal. Incubate in a 37°C water bath for 2–10 minutes; the precipitate will usually dissolve completely.
2. For cell proliferation assays, seed 2000 cells in 100 μL per well.For cytotoxicity assays, seed 5000 cells in 100 μL per well.The exact cell number per well should be determined according to cell size, proliferation rate, and other factors.Culture cells and treat with 0–10 μL of the specified drug as required.
3. Add 10 μL of WST‑1 solution to each well.If the initial culture volume is 200 μL, add 20 μL of WST‑1 solution, and scale proportionally for other volumes.Wells containing the corresponding volume of cell culture medium and WST‑1 solution but no cells can be used as blank controls.
4. Incubate for 0.5–4 hours in a cell culture incubator; 1–2 hours is sufficient for most cell types.The incubation time depends on cell type and density.For initial experiments, measure absorbance at 0.5, 1, 2, and 4 hours using a microplate reader, then select the time point with an appropriate absorbance range for subsequent experiments.
5. Place the 96‑well plate on an orbital shaker and shake for one minute to fully mix the assay system.
6. Measure absorbance at 450 nm.If a 450 nm filter is unavailable, a filter within 420–480 nm can be used.A wavelength above 600 nm can be used as the reference wavelength for dual‑wavelength measurement.
Precautions
1. As the assay is performed using a 96‑well plate, evaporation must be considered during long‑term cell culture.On one hand, the outer wells of the plate are most prone to evaporation; these can be left unused and filled with an equal volume of PBS, water, or medium.On the other hand, placing the plate near the water source inside the incubator can help reduce evaporation.
2. The detection in this kit relies on dehydrogenase‑catalyzed reactions.Therefore, reducing agents (such as certain antioxidants) may interfere with the assay.If a high level of reducing agents is present in the test system, they should be removed beforehand.
3. Ensure there are no air bubbles in any well before measurement with a microplate reader, as bubbles will interfere with the absorbance reading.
4. This product is for research use only by qualified personnel.It is not intended for clinical diagnosis or treatment, or for use in food or drug products.Do not store in ordinary residential premises.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
ZJ26F0433906
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