超氧化物检测试剂盒
产品编号: S0060
产品包装:100次
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超氧化物检测试剂盒(S0060)
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价格: ¥ 558.00
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| 产品编号 | 产品名称 | 产品包装 | 产品价格 |
| S0060 | 超氧化物检测试剂盒 | 100次 | 558.00元 |
超氧化物检测试剂盒(Superoxide Assay Kit)是一种用于超氧化物快速高灵敏度检测的试剂盒。
本试剂盒利用超氧化物可以还原WST-1产生可溶性有色物质为基础来检测超氧化物。本试剂盒的检测试剂中添加了Catalase(触酶)等，可以清除过氧化氢等过氧化物对WST-1显色的干扰，使测定结果更加准确。并且本试剂盒还提供SOD(超氧化物歧化酶)，可以验证本试剂盒测定出的结果是否为超氧化物，以排除检测体系中所用的一些试剂可能产生的干扰。对于本试剂盒，加入SOD后通常可以抑制90%以上的超氧化物。
WST-1是一种类似于MTT的化合物，可以被超氧化物还原生成橙色的formazan。超氧化物产生越多越快，则颜色越深，即相应测定得到的吸光度值越大。
使用WST-1作为检测试剂比用传统的细胞色素c(cytochrome c)检测超氧化物具有多方面的优点。首先，WST-1本身的吸光度非常低(通常OD450为0.02左右，因仪器和酶标板不同而有所不同)，而细胞色素c本身的吸光度非常高(通常OD550为0.5左右)，因此，使用WST-1与细胞色素c相比，检测灵敏度高很多倍。其次，WST-1的还原产物是稳定的可溶性产物，且对细胞基本无毒性，使WST-1方法更加适合于高通量的筛选。另外，细胞色素c法由于灵敏度的限制，一般不适合用96孔板测定，而WST-1法可以用96孔板测定，使检测更加便捷。用WST-1法或细胞色素c法对超氧化物测定效果比较参考图1。图1中，50万/毫升嗜中性粒细胞在37℃用PMA刺激指定时间，用WST-1或细胞色素c方法分别测定。WST-1法测定OD450，而细胞色素c法测定OD550。另外，WST-1法和luminol法相比，灵敏度相近，但仅须使用普通的酶标仪，无须使用luminol法所需的luminometer，并且检测速度也比luminol法更加快捷。
超氧化物通常指超氧化物阴离子(superoxide anion)O2.-，是一种氧分子的自由基。在呼吸链中，NADPH氧化酶把电子传递给氧分子的时候，就会产生超氧化物阴离子O2.-。超氧化物阴离子O2.-是一种强氧化剂，可以由被刺激的白细胞等产生，从而抵御微生物的感染等。超氧化物阴离子O2.-也可以导致氧化损伤，和许多疾病的发生密切相关。
本试剂盒可以测定100个样品。
图1. WST-1法和细胞色素c法对于超氧化物测定效果的比较。
包装清单：
| 产品编号 | 产品名称 | 包装 |
| S0060-1 | 超氧化物检测缓冲液 | 30ml |
| S0060-2 | WST-1溶液 | 1ml |
| S0060-3 | Catalase溶液 | 200μl |
| S0060-4 | SOD溶液 | 20μl |
| — | 说明书 | 1份 |
保存条件：
-20℃保存，一年有效。
注意事项：
WST-1溶液、Catalase溶液和SOD溶液均宜尽量避免反复冻融，可以适当分装。
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。
为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：
1. 超氧化物检测工作液的配制：
按照下表比例配制超氧化物检测工作液：
| 1个检测 | 5个检测 | 10个检测 | 20个检测 | 50个检测 | |
| 超氧化物检测缓冲液 | 200微升 | 1毫升 | 2毫升 | 4毫升 | 10毫升 |
| WST-1溶液 | 10微升 | 50微升 | 100微升 | 200微升 | 500微升 |
| Catalase溶液 | 2微升 | 10微升 | 20微升 | 40微升 | 100微升 |
| 超氧化物检测工作液 | 212微升 | 1.06毫升 | 2.12毫升 | 4.24毫升 | 10.6毫升 |
2. 悬浮细胞产生超氧化物的检测：
a. 细胞用PBS、Hanks液或生理盐水洗涤一次。
b. 约按5-10万个细胞/毫升的比例，用超氧化物检测工作液悬浮细胞。
c. 在96孔板中，每孔加入200微升用超氧化物检测工作液悬浮的细胞，每孔约1-2万个细胞。
d. 37℃孵育3分钟。
e. 在预定的检测孔中加入预计可以诱导产生超氧化物的刺激物，如PMA等，刺激10-60分钟或其它预定的时间。须至少留一个不加刺激物的孔作为空白对照。选择1-2个加刺激物的孔中加入2微升SOD以验证整个检测体系，加SOD溶液的检测可以不做。试剂盒提供的SOD溶液足够做10个检测。具体检测时的安排参考下表：
| 细胞 | 超氧化物检测工作液 | 刺激物 | SOD溶液 | |
| 空白对照 | ＋ | ＋ | － | － |
| 待测样品 | ＋ | ＋ | ＋ | － |
| 阴性对照 | ＋ | ＋ | ＋ | ＋ |
f. 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。
3. 贴壁细胞产生超氧化物的检测：
a. 约按每孔5000-10000个细胞(具体的细胞接种数量须根据细胞的大小和生长速度自行确定)把细胞培养到96孔板中，使第二天或待检测时细胞为80-90%满。
b. 吸去培养液，用PBS、Hanks液或生理盐水洗涤一次。
c. 每孔加入200微升超氧化物检测工作液，37℃孵育3分钟。
d. 在预定的检测孔中加入预计可以诱导产生超氧化物的刺激物，如PMA等，刺激10-60分钟或其它预定的时间。须至少留一个不加刺激物的孔作为空白对照。选择1-2个加刺激物的孔中加入2微升SOD以验证整个检测体系，加SOD溶液的检测可以不做。试剂盒提供的SOD溶液足够做10个检测。具体检测时的安排参考下表：
| 细胞 | 超氧化物检测工作液 | 刺激物 | SOD溶液 | |
| 空白对照 | ＋ | ＋ | － | － |
| 待测样品 | ＋ | ＋ | ＋ | － |
| 阴性对照 | ＋ | ＋ | ＋ | ＋ |
e. 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。
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