DCFH-DA 本身没有荧光,可以自由穿过细胞膜,进入细胞内后,可以被细胞内的酯酶水解生成DCFH。而 DCFH 不能通透细胞膜,从而使探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH 生成有荧光的DCF 。检测DCF 的荧光就可以知道细胞内活性氧的水平。
操作步骤(仅供参考):
一、制备探针:10mM(4.8729mg溶于1mL DMSO)
二、装载探针:
对于刺激时间较短(通常为2 小时以内)的细胞,先装载探针,后用活性氧阳性对照或自己感兴趣的药物刺激细胞。对于细胞刺激时间较长(通常为6 小时以上)的细胞,先用活性氧阳性对照或自己感兴趣的药物刺激细胞,后装载探针。
原位装载探针:本方法仅适用于贴壁培养细胞。按照1 ∶1000用无血清培养液稀释DCFH-DA,使终浓度为10μ mol/L。去除细胞培养液,加入适当体积稀释好的DCFH-DA。加入的体积以能充分盖住细胞为宜,通常对于六孔板的一个孔加入稀释好的DCFH-DA不少于1mL 。37℃细胞培养箱内孵育20 分钟。用无血清细胞培养液洗涤细胞三次,以充分去除未进入细胞内的DCFH-DA。通常活性氧阳性对照在刺激细胞20~30分钟后可以显著提高活性氧水平。
收集细胞后装载探针:按照1 ∶1000 用无血清培养液稀释DCFH-DA,使终浓度为10μmol/L。细胞收集后悬浮于稀释好的DCFH-DA中,细胞浓度为一百万至二千万/mL ,37℃细胞培养箱内孵育20分钟。每隔3 ~5 分钟颠倒混匀一下,使探针和细胞充分接触。用无血清细胞培养液洗涤细胞三次,以充分去除未进入细胞内的DCFH-DA。直接用活性氧阳性对照或自己感兴趣的药物刺激细胞,或把细胞等分成若干份后刺激细胞。通常活性氧阳性对照在刺激细胞20~30分钟后可以显著提高活性氧水平。
三、检测:
对于原位装载探针的样品可以用激光共聚焦显微镜直接观察,或收集细胞后用荧光分光光度计、荧光酶标仪或流式细胞仪检测。对于收集细胞后装载探针的样品可以用荧光分光光度计、荧光酶标仪或流式细胞仪检测,也可以用激光共聚焦显微镜直接观察。
四、参数设置
使用488nm 激发波长,525nm 发射波长,实时或逐时间点检测刺激前后荧光的强弱。DCF 的荧光光谱和FITC非常相似,可以用FITC的参数设置检测DCF 。
注意事项:
1. 探针装载后,一定要洗净残余的未进入细胞内的探针,否则会导致背景较高。
2. 探针装载完毕并洗净残余探针后,可以进行激发波长的扫描和发射波长的扫描,以确认探针的装载情况是否良好。DCF 的激发光谱和发射光谱请参考上图。
3. 尽量缩短探针装载后到测定所用的时间(刺激时间除外),以减少各种可能的误差。
4. 对于某些细胞,如果发现没有刺激的阴性对照细胞荧光也比较强,可以按照1 ∶2000~1 ∶5000稀释DCFH-DA,使装载探针时DCFH-DA的浓度为2~5μmol/L。探针装载的时间也可以根据情况在15~60分钟内适当进行调整。
5. 为了您的安全和健康,请穿实验服并戴一次性手套操作。
使用本产品的应用案例(仅供参考)
In Vitro
Cell(HK-2 human kidney proximal tubular cells,20 μmol/L DCFH-DA,37℃ for 30 min)
Cells grown on coverslips were incubated with 20 μmol/L 2′,7′-dichlorofluorescin diacetate at 37 ◦C for 30 min in the dark and then were washed with phosphate buffer saline. Cells were fixed in 4% paraformaldehyde, counterstained with DAPI, then washed 3 times with phosphate buffer saline. Cells were mounted with an anti-fading mounting medium and fluorescence intensity was detected by a fluorescence microscope with an excitation wavelength at 488 nm and an emission wavelength at 525 nm.
来源文献:Li S, Zheng L, Zhang J, Liu X, Wu Z. Inhibition of ferroptosis by up-regulating Nrf2 delayed the progression of diabetic nephropathy. Free Radic Biol Med. 2021 Jan;162:435-449. doi: 10.1016/j.freeradbiomed.2020.10.323. Epub 2020 Nov 2. PMID: 33152439.
Cell(vascular smooth muscle cells/VSMCs; 10 μM DCFH- DA; 37℃,30 min)
The intracellular ROS content was measured with the fluorescent probe DCFH- DA(Solarbio, China), which itself is nonfluorescent. After freely passing through the cell membrane, Tt is hydrolyzed to impenetrable DCFH and remains inside the cells. Intracellular ROS oxidizes DCFH to fluorescent DCF, which indicates the intracellular ROS levels according to the fluorescence intensity. VSMCs with five different treatments were incubated with serum-free DMEM with a DCFH-DA final concentration of 10 uM for 30 min at 37 ◦C. Next, we washed the VSMC-DCFH-DA mixture with serum-free DMEM three times and resuspended it in 1× PBS solution. The prepared VSMC samples were analyzed using flow cytometry.
来源文献:Shao S, Liu Y, Hong W, Mo Y, Shu F, Jiang L, Tan N. Influence of FOSL1 Inhibition on Vascular Calcification and ROS Generation through Ferroptosis via P53-SLC7A11 Axis. Biomedicines. 2023 Feb 20;11(2):635. doi: 10.3390/biomedicines11020635. PMID: 36831172; PMCID: PMC9953509.
Cell(myoblasts;10 μM DCFH-DA,37°C,20 min)
The myoblasts were cultured in the 35 mm cell culture dishes, and after heat stress, the cells were incubated with 10 μM dichlorofluorescein diacetate (DCFH-DA) (Solarbio, Beijing,China) at 37°C for 20 min, after washing three times by DMEM/F12 to remove the extracellular DCFH-DA of myoblasts, the laser scanning confocal microscope was used to obtain the ROS images.
来源文献:Lu J, Li H, Yu D, Zhao P, Liu Y. Heat stress inhibits the proliferation and differentiation of myoblasts and is associated with damage to mitochondria. Front Cell Dev Biol. 2023 Apr 11;11:1171506. doi: 10.3389/fcell.2023.1171506. PMID: 37113771; PMCID: PMC10126414.
Cell(PC12 cells;10 μM DCFH-DA ;37 °C,10 min)
The cells (1×104/well) were seeded in 96-well plates, and were treated with various concentrations of baicalein (40, 60 and 80 μM) for 2 h, followed by incubation with Aβ25–35 (20 μM) for 24 h. Then, the cells were incubated with 10 μM DCFH-DA and 10 μM Hoechst 33342 for 10 min at 37 °C in the darkness, and washed three times with PBS. The fluorescence intensity was detected and the images were photographed using a Cytation imaging reader. The intracellular ROS levels were indicated by total fluorescence intensity.
来源文献:Gao L, Zhou F, Wang KX, Zhou YZ, Du GH, Qin XM. Baicalein protects PC12 cells from Aβ25-35-induced cytotoxicity via inhibition of apoptosis and metabolic disorders. Life Sci. 2020 May 1;248:117471. doi: 10.1016/j.lfs.2020.117471. Epub 2020 Feb 26. PMID: 32112868.
Cell(NRK-52E cells)
NRK-52E cells were first seeded in six-well plates at a density of 1×105 cells per well and cultured for 24 h at 37℃. Then, the cells were stimulated by DMEM high glucose and incubated with different concentrations of CSMDNPs for 12 h. Subsequently, the redox-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Solarbio) was used for the monitoring of intracellular levels of oxidative stress, and the reactive free radicals labeled with green fluorescence were observed using fluorescence microscope .
来源文献:Sun J, Han J, Dong J, Zhai X, Zhang R. A kidney-targeted chitosan-melanin nanoplatform for alleviating diabetic nephropathy through modulation of blood glucose and oxidative stress. Int J Biol Macromol. 2024 Apr;264(Pt 2):130663. doi: 10.1016/j.ijbiomac.2024.130663. Epub 2024 Mar 6. PMID: 38453104.
Cell(4T1 cells,6 h later, the cells were washed with PBS for three times and stained with DCFH-DA for another 30 min.)
2’, 7’-dichlorofluorescein diacetate (DCFH-DA) was employed as the indicator to investigate the intracellular ROS generation. Briefly, 4T1 cells (1 × 104) were seeded in a 96-well plate and cultured for 24 h. Then the HPCOF solution (200 μg/mL) was added. 6 h later, the cells were washed with PBS for three times and stained with DCFH-DA for another 30 min. After that, the cells were washed by PBS for three times to remove the excessive DCFH-DA. Finally, the cells were irradiated by a 660 nm laser for 5 min. Intracellular ROS generation were observed by using a fluorescence microscope.
来源文献:Liu Y , Yang K , Wang J ,et al.Hypoxia-triggered degradable porphyrinic covalent organic framework for synergetic photodynamic and photothermal therapy of cancer[J].Materials Today Bio, 2024, 25.DOI:10.1016/j.mtbio.2024.100981.
Cell(Plant somatic embryogenesis (SE))
An ROS-specific fluorescent probe H2DCF (Molecular Probes, ID3130; Solarbio, Beijing, China) was used to assess intracellular ROS levels.
来源文献:Yuan J, Liu X, Zhao H, Wang Y, Wei X, Wang P, Zhan J, Liu L, Li F, Ge X. GhRCD1 regulates cotton somatic embryogenesis by modulating the GhMYC3-GhMYB44-GhLBD18 transcriptional cascade. New Phytol. 2023 Oct;240(1):207-223. doi: 10.1111/nph.19120. Epub 2023 Jul 11. PMID: 37434324.
These protocols are for reference only. Solarbio does not independently validate these
methods.
你好,我想问一下如果是拍倒置荧光的话。需要分散在培养基里面还是PBS里亚?2023-06-06 11:24:48
您好,细胞分散在PBS或无血清培养基中均可。
DMSO溶解好的储存液-20度能储存多久?2022-10-10 10:43:25
您好,-20℃配制好的储备液可以保存一个月。