Hoechst 33342
(Synonyms: 赫斯特荧光染料33342,bisBenzimide H 33342; HOE 33342) 目录号 : GC63515
核酸染色剂Hoechst 33342 (Ex/Em: 350/461 nm) 常被用作细胞可渗透的核染色剂,与双链DNA结合后发出蓝色荧光。
Cas No.:23491-52-3
Sample solution is provided at 25 µL, 10mM.
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Related Biological Data
PAD@MS improves tumor immunogenicity to activate immune response. B) CLSM imaging of CRT on the surface of MC38 tumor cells after treatments.
Then, cells were washed with PBS, incubated with Alexa Fluor 488-conjugated anti-CRT for 1 h, stained with Hoechst 33342 (GlpBio, USA), ER Tracker, or Dil for 10 min, and observed under CLSM.
Adv Funct Mater 33.35 (2023): 2214998. PMID: 35724919 IF: 19.9246 -
Related Biological Data
Rhamnose interacts with SLC12A4 protein. (B) Representative confocal images of Bio-Rhamnose (Bio-Rha) binding to the surface of BMDMs. Confocal image of cells labeled with probes (red) and Hoechst 33342 stain (blue).
After washing with PBS for three times, the cell membrane was labeled with Wheat germ agglutinin, for 30 min,and cell nucleus was labeled with Hoechst 33342 (Cat# GC63515, GlpBio, USA) for 10 min.
Acta Pharm Sin B (2024). IF: 14.5
Quality Control & SDS
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- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1、制备Hoechst 33342染色液
(1) 配制Hoechst 33342染料储存液: 使用DMSO溶解固体Hoechst 33342，配置成10mg/mL的Hoechst 33342染料储存液。
注意: Hoechst储存液建议分装后于-4℃或-20℃避光保存，避免反复冻融。
(2)工作液制备:使用预热的无血清培养基或缓冲液(如HBSS或PBS)稀释储存液，配制浓度为10μg/mL的Hoechst 33342工作液。
注意: 请根据实际情况调整 Hoechst 工作液浓度，现用现配。
2、细胞染色
2.1 悬浮细胞（以6孔板为例）
(1)悬浮细胞经1000g离心3-5min。弃去上清液，使用PBS清洗两次，每次5分钟。
(2)加入1mL的Hoechst 33342染料工作液，室温避光孵育5-10 min分钟。
(3)孵育结束后，经1000g离心5分钟，去除上清液，加入PBS清洗2-3次，每次5分钟。
(4)使用无血清细胞培养基或PBS重悬细胞，通过荧光显微镜或流式细胞技术进行观察。
2.2 贴壁细胞
(1)在无菌盖玻片上培养贴壁细胞。
(2)从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中。
(3)从盖玻片的一角加入100uL的Hoechst 33342染料工作液，轻轻晃动使染料均匀覆盖所有细胞，室温避光孵育5-15min分钟。
(4)吸弃染料工作液，使用培养液洗盖玻片2~3次，通过荧光显微镜进行观察。
3.显微镜检测：Hoechst 33342的激发/发射光分别为350/460nm。
注意事项：
①对于固定的细胞或组织样品的染色，固定后需漂洗去除固定剂；
②Hoechst 33342染色通常在其他染色后进行，如果不需要进行其它染色，则直接进行Hoechst 33342染色；
③为减缓荧光淬灭，建议使用抗荧光淬灭封片剂；
④荧光染料均存在淬灭问题，请尽量注意避光；
⑤Hoechst 33342对人体有一定刺激性，为了您的安全和健康，请穿实验服并戴一次性手套操作。
References:
[1]. Lisa C Crowley, Brooke J Marfell , Nigel J Waterhouse. Analyzing Cell Death by Nuclear Staining with Hoechst 33342. 2016 Sep 1;2016(9). doi: 10.1101/pdb.prot087205.
The nucleic acid stain Hoechst 33342 (Ex/Em: 350/461 nm) is frequently utilized as a cell-permeable nuclear counterstain that emits a blue fluorescence upon binding to dsDNA. Hoechst 33342 is commonly employed in various studies related to cell counting, cell cycle analysis, and cell replication. It is particularly useful in identifying condensed nuclei in apoptotic cells, as well as in combination with BrdU staining for cell-cycle studies.
Hoechst dyes are also useful for monitoring cell viability by tracking changes in their emission spectra. As minor groove-binding DNA stains with AT selectivity, the Hoechst dyes are able to bind to all nucleic acids, but they show a greater fluorescence enhancement for AT-rich double-stranded DNA strands compared to GC-rich strands [1]. This property has been exploited to identify Q-bands in chromosomes, which are regions rich in AT base pairs that fluoresce brightly when stained with the quinacrine dye [2].
Fig. Fluorescence excitation and emission spectra of Hoechst 33342 bound to DNA
References:
[1]. Portugal J, Waring MJ. Assignment of DNA binding sites for 4′, 6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression. 1988 Feb 28;949(2):158-68.
[2]. Weisblum B, Haenssler E. Fluorometric properties of the bibenzimidazole derivative Hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA. Chromosoma. 1974 Sep;46(3):255-60.
核酸染色剂Hoechst 33342 (Ex/Em: 350/461 nm) 常被用作细胞可渗透的核染色剂,与双链DNA结合后发出蓝色荧光。Hoechst 33342常被用于与细胞计数、细胞周期分析和细胞复制相关的各种研究中。它特别有用于鉴定凋亡细胞中的紧缩细胞核,并与BrdU染色结合在一起进行细胞周期研究。
Hoechst染料还可通过跟踪其发射光谱的变化来监测细胞活力。作为小沟结合DNA染料,具有AT选择性,Hoechst染料能够结合所有核酸,但与GC富集的双链DNA链相比,它们对富含AT碱基的链显示出更大的荧光增强[1]。这种特性已被利用于鉴定染色体中的Q带,这些区域富含AT碱基对,当用quinacrine染料染色时会发出明亮的荧光[2]。
| Cas No. | 23491-52-3 | SDF | |
| 别名 | 赫斯特荧光染料33342,bisBenzimide H 33342; HOE 33342 | ||
| 分子式 | C27H28N6O | 分子量 | 452.55 |
| 溶解度 | DMSO : 10 mg/mL (22.10 mM; Need ultrasonic)|Water : 1 mg/mL (2.21 mM; ultrasonic and warming and heat to 80°C) | 储存条件 | 4°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.2097 mL | 11.0485 mL | 22.097 mL |
| 5 mM | 0.4419 mL | 2.2097 mL | 4.4194 mL |
| 10 mM | 0.221 mL | 1.1049 mL | 2.2097 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Analyzing Cell Death by Nuclear Staining with Hoechst 33342
Cold Spring Harb Protoc 2016 Sep 1;2016(9).PMID:27587774DOI:10.1101/pdb.prot087205.
The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes.
Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy
Photochem Photobiol Sci 2010 Dec;9(12):1634-9.PMID:20931137DOI:10.1039/c0pp00234h.
Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy. A caveat is that the probes themselves should not perturb cellular responses and under normal conditions the dyes are generally non-toxic. However, researchers are increasingly using computerized time-lapse microscopy (CTLM), where cells stained with fluorescent dyes are often imaged frequently over a period of several days, to follow cellular responses in real time. Little is currently known about possible toxicity of fluorescent DNA dyes under CTLM conditions. In this study we demonstrate that the common live-cell DNA stain Hoechst 33342 can cause apoptosis under CTLM conditions. Although toxicity is evident at long times in the absence of imaging at high dye concentrations, phototoxicity from repeated excitation of the dye in the imaging process is dominant. We show that phototoxicity is a function of the product of light fluence and dye concentration, irrespective of irradiance, frequency and total number of scans. Thus, phototoxicity can be prevented by a combination of dye concentration and imaging procedure that is below this threshold. These quantitative data can be used as a guide to others performing time-lapse microscopy studies with this common live-cell DNA stain and serves as a caution for researchers when using other fluorescent stains under CTLM conditions.
Flow Cytometric Detection of G0 in Live Cells by Hoechst 33342 and Pyronin Y Staining
Methods Mol Biol 2018;1686:49-57.PMID:29030811DOI:10.1007/978-1-4939-7371-2_3.
Hoechst 33342 and Pyronin Y double staining can be used to measure DNA and RNA content in live cells by flow cytometry. Quiescent cells at G0 phase have the same amount of DNA as cells at G1 phase but lower RNA levels compared to proliferating cells. Therefore, resting cells in G0 phase can be distinguished from proliferating cells in G1, S, and G2 M phases. This chapter describes a protocol for double staining of live cells with Hoechst 33342 and Pyronin Y. Combined with immunophenotyping of intact and live cells Hoechst 33342 and Pyronin Y staining is a powerful noninvasive method for the analysis and isolation of quiescent cells from any defined cell population.
Hoechst 33342 as a marker for imaging neurites of Dorsal Root Ganglion in vitro
J Anat 2022 May;240(5):998-1001.PMID:PMC9005665DOI:10.1111/joa.13599.
Fluorescent markers, generally targeting neurotubules, are used to visualize the radiating crown of growing neurites that is produced by dorsal root ganglion cells in vitro. Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate) is a widely used fluorescent DNA marker that stain both live and fixed nuclei. We have recently found that H33342 can visualize the neurites of DRG too, but only when they are fixed in formalin. Images have a good signal-to-noise ratio. We noticed that besides H33342 being a specific marker for DNA, it also stains the transmembrane P-glycoprotein (P-gp) which is involved in the active pump-out of alien molecules from the cytoplasm; so, H33342 remains associated with P-gp after fixation. P-gp is quite ubiquitous in healthy cells and, notably, P-gp has been detected in DRG of several species as well as in human DRG. The use of H33342 as a staining for neurites of DRG in fixed samples could have a practical value due to its widespread use and its better affordability compared to other fluorescent markers for neurites.
Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP
PLoS One 2015 Nov 5;10(11):e0141991.PMID:26540112DOI:10.1371/journal.pone.0141991.
Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden "Janus" amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of "Hoechst 33342-like" drugs to cells and tissues in which these transporters are expressed.