Reactive Oxygen Species (ROS) Assay Kit 活性氧（ROS）檢測試劑盒

搜索關鍵詞：

Reactive Oxygen Species，ROS活性氧；DCFH-DA；JC-1線粒體模電(diàn)位探針；JC-10；CAS NO：4091-99-0；

産品訂購：

研究意義：

活性氧（Reactive Oxygen Species，ROS）包括超氧自(zì)由基、過氧化氫、及其下(xià)遊産物(wù)過氧化物(wù)和羟化物(wù)等，參與細胞生(shēng)長(cháng)增殖、發育分化、衰老和凋亡以及許多(duō)生(shēng)理和病理過程。

産品描述：

活性氧檢測試劑盒（Reactive Oxygen Species Assay Kit，或ROS Assay Kit）是一(yī)種利用熒光(guāng)染料DCFH-DA（2,7-Dichlorodihydrofluorescein diacetate）進行細胞内活性氧水(shuǐ)平檢測的常用方法。檢測原理在于DCFH-DA本身無熒光(guāng)，能(néng)自(zì)由穿過細胞膜，一(yī)旦進入細胞後，被胞内的酯酶水(shuǐ)解生(shēng)成無熒光(guāng)的DCFH。DCFH不能(néng)穿透細胞膜，從(cóng)而保留在胞内。此時胞内的活性氧可以氧化DCFH生(shēng)成有熒光(guāng)的DCF。通(tōng)過DCF熒光(guāng)的強弱即可反映活性氧的水(shuǐ)平。

本試劑盒包含活性氧陽性對照(zhào)Rosup，利于活性氧的檢測。Rosup是一(yī)種混合物(wù)，濃度為(wèi)50mg/ml。因檢測對象和反應體系的不同，本試劑盒可檢測100-500個(gè)樣品。

産品組成：

保存與運輸方法：

保存：-20ºC保存，有效期一(yī)年(nián)。

運輸：冰袋運輸。

使用方法

1. 裝載探針

【注意】：對于刺激時間較短（通(tōng)常2h以内）的細胞，先裝載探針，後用活性氧陽性對照(zhào)Rosup和/或待研究藥物(wù)刺激細胞；對于刺激時間較長(cháng)（通(tōng)常6h以上(shàng)）的細胞，先用活性氧陽性對照(zhào)Rosup或待研究藥物(wù)刺激細胞，後裝載探針。

1.1 原位裝載探針（僅适用于貼壁細胞）

1）檢測前一(yī)天進行細胞鋪闆，确保活性氧檢測當天細胞彙合率達到(dào)50~70%。

2）吸出細胞培養液，加入适量體積以及濃度的待研究藥物(wù)，于37ºC細胞培養箱内孵育，具體誘導時間根據細胞類型和藥物(wù)自(zì)身特性決定。

【可選】陽性對照(zhào)Rosup：先用無血清培養液按照(zhào)1:1000比例稀釋陽性對照(zhào)，通(tōng)常37℃培養箱内刺激20~30min可以觀察到(dào)顯著的活性氧水(shuǐ)平提高(gāo)，但依細胞類型會(huì)有比較明顯差異【刺激時間可參考文獻或實驗經驗來調整】。如果刺激後30min内未觀察到(dào)活性氧水(shuǐ)平升高(gāo)，可以适當提高(gāo)Rosup的工(gōng)作濃度；反之，如果活性氧升高(gāo)過快，則适當降低(dī)Rosup的工(gōng)作濃度。

3）按照(zhào)1:1000用無血清培養液稀釋DCFH-DA（10mM），使其終濃度為(wèi)10μM.。吸出細胞培養液，加入适當體積DCFH-DA工(gōng)作液。【注意】：加入體積以能(néng)充分蓋住細胞為(wèi)宜。對于6孔闆，每個(gè)孔加入DCFH-DA工(gōng)作液不少于1ml。對于96孔闆，每個(gè)孔加入DCFH-DA工(gōng)作液不少于0.1ml。

4）37ºC細胞培養箱内孵育20min。

5）用無血清細胞培養液洗滌細胞三次，以充分去除未進入細胞内的DCFH-DA。

1.2 收集細胞後裝載探針

1）按照(zhào)常規方法，清洗并收集足量的細胞。【注意】：保證細胞的狀态健康。

2）用适量體積以及濃度的待研究藥物(wù)重新懸浮細胞，于37ºC細胞培養箱内孵育，具體誘導時間根據細胞類型和藥物(wù)自(zì)身特性決定。【可選】陽性對照(zhào)Rosup：先用無血清培養液按照(zhào)1:1000比例稀釋陽性對照(zhào)，通(tōng)常37℃培養箱内刺激20~30min可以觀察到(dào)顯著的活性氧水(shuǐ)平提高(gāo)，但依細胞類型會(huì)有比較明顯差異【刺激時間可參考文獻或實驗經驗來調整】。如果刺激後30min内未觀察到(dào)活性氧水(shuǐ)平升高(gāo)，可以适當提高(gāo)Rosup的工(gōng)作濃度；反之，如果活性氧升高(gāo)過快，則适當降低(dī)Rosup的工(gōng)作濃度。

3）探針裝載前，按照(zhào)1:1000用無血清培養液稀釋DCFH-DA（10mM），使其終濃度為(wèi)10μM.。

4）離心，吸出細胞内刺激藥物(wù)，之後用适量DCFH-DA工(gōng)作液重懸細胞，使得細胞密度為(wèi)1×106~2×107/ml。【注意】：細胞密度需根據後續檢測體系，檢測方法，以及檢測總量來調整。例如，對于流式分析，單管檢測内細胞數目控制在104~ 106之間。

5）37ºC細胞培養箱内孵育20min。每隔3~5min颠倒混勻一(yī)下(xià)，使探針和細胞充分接觸。

6）用無血清細胞培養液洗滌細胞三次，以充分去除未進入細胞内的DCFH-DA。

2. 檢測

2.1 原位裝載法

可用激光(guāng)共聚焦顯微鏡直接觀察，或收集細胞後用熒光(guāng)分光(guāng)光(guāng)度計、熒光(guāng)酶标儀或流式細胞儀檢測。

2.2 收集細胞後裝載法

可用熒光(guāng)分光(guāng)光(guāng)度計、熒光(guāng)酶标儀或流式細胞儀檢測，也可用激光(guāng)共聚焦顯微鏡直接觀察。

3. 參數設置

使用488nm激發波長(cháng)，525nm發射波長(cháng)，實時或逐時間點檢測刺激前後熒光(guāng)的強弱。DCF的熒光(guāng)光(guāng)譜和FITC非常相(xiàng)似，可用FITC的參數設置檢測DCF。

4. 其他說明

1）對于某些細胞，若發現沒有刺激的陰性對照(zhào)細胞熒光(guāng)也比較強，可以按照(zhào)1:2000～1:5000稀釋DCFH-DA，使DCFH-DA的工(gōng)作濃度為(wèi)2～5μM。探針裝載時間也可依情況在15～60min内适當進行調整。

2）Rosup僅僅用于作為(wèi)陽性對照(zhào)的樣品，并不是在每個(gè)樣品中都需加入Rosup陽性對照(zhào)。

注意事(shì)項

1）探針裝載後，一(yī)定要洗淨殘餘的未進入細胞内的探針，否則會(huì)導緻背景較高(gāo)。

2）探針裝載完畢并洗淨殘餘探針後，可以進行激發波長(cháng)的掃描和發射波長(cháng)的掃描，以确認探針的裝載情況是否良好。

3）盡量縮短探針裝載後到(dào)測定所用的時間（刺激時間除外），以減少各種可能(néng)的誤差。

4）為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

 — — Written/Edited by V. Shallan【版權歸MKBio懋康所有】

上海夢澤生物科技有限公司是一(yī)家涉足于生(shēng)命科學和生(shēng)物(wù)技(jì)術(shù)領域研究的試劑、儀器(qì)和實驗室消耗品與實驗服務工(gōng)作，主要從(cóng)事(shì)細胞生(shēng)物(wù)學、植物(wù)學、分子生(shēng)物(wù)學、免疫學、生(shēng)物(wù)化學、蛋白(bái)組學。生(shēng)物(wù)制藥與診斷試劑研發生(shēng)産等領域。 本公司秉承“以人為(wèi)本，以誠為(wèi)信、合同守信”的經營理念。堅持"品質保障"的原則為(wèi)廣大客戶提供優質産品。

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HEK293 cells were first transfected with pCAGGS and pCAGGS-PRDX1. When PRDX1 was highly expressed for about 24 h, cells were infected with DTMUV and treated for 6 h, whereas the Rosup positive control group was set in parallel. Finally, the probe of DCFH-DA was diluted at 1:1000 and treated at 30 °C for 20 min referring to the ROS Assay Kit (MKBio, Shanghai, China). Cells were then observed under a fluorescent inverted microscope immediately.

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The DCFH-DA ROS assay kit (MKBio, Shanghai, China) was used to measure the ROS levels of the GC cells. In brief, GC cells were prepared and incubated with 5 μM DCFH-DA staining solution in the dark for 30 min at 37 °C.

The DCFH-DA ROS assay kit (MKBio, Shanghai, China) was used to measure the ROS levels of the GC cells. In brief, GC cells were prepared and incubated with 5 μM DCFH-DA staining solution in the dark for 30 min at 37 °C.

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The ROS production in the seedlings was detected by using fluorescent dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) (Maokang, Shanghai, China), as described previously [32]. The 7-day-old different-genotype seedlings grown on MS solid medium were exposed to 45 °C for 3 h, and the seedlings were collected and stained at 22 °C for 15 min in 50-μM H2DCFDA (dissolved in MES-KCl buffer, MES 10 mM, KCl 50 mM, adjust to pH 5.5 with HCl). The seedlings washed with distilled water were placed under a Leica confocal microscope. The fluorescence in cotyledons and roots of seedlings were detected by using an excitation wavelength of 488 nm

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Shen LD, Qi WH, Bai JJ, Zuo CY, Bai DL, Gao WD, Zong XL, Hao TT, Ma Y, Cao GC. Resibufogenin inhibited colorectal cancer cell growth and tumorigenesis through triggering ferroptosis and ROS production mediated by GPX4 inactivation. Anat Rec (Hoboken). 2021 Feb;304(2):313-322. doi: 10.1002/ar.24378. Epub 2020 Feb 7. PMID: 31961485.

The intracellular ROS levels in CRC cells were measured by using the
2,7-dichlorodihydrofluorescein diacetate (DCFH- DA) ROS assay kit purchased from MKBio
(Shanghai, China) according to the manufacturer's instruction 

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The astrocytes were planted on 24-well cell culture plates at a density of 1 × 10 5 cells/well for 24 h. After necessary stimulation, cells were loaded with 5 μM DCFH-DA (MX4802, MKBio, China) and incubated for 30 min in darkness.