Hoechst 33342

Manufactured by Beyotime

Sourced in China, United States, Japan, Germany

Hoechst 33342 is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in cell biology and microscopy applications to stain and visualize cell nuclei.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (30 mentions) Fluorescence microscope, by Olympus (25 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (17 mentions) Lsm 880, by Zeiss (16 mentions) Lsm 710, by Zeiss (14 mentions) Lyso tracker red, by Beyotime (13 mentions) Fluorescence microscope, by Leica (13 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions) Mito tracker green, by Beyotime (9 mentions) Mito tracker red cmxros, by Beyotime (9 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (9 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions) Axio observer z1, by Zeiss (9 mentions) Mitosox red, by Thermo Fisher Scientific (8 mentions) Propidium iodide, by Beyotime (8 mentions) Lysotracker green, by Beyotime (7 mentions) Bovine serum albumin (bsa), by Merck Group (7 mentions) Sytox green, by Thermo Fisher Scientific (7 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (6 mentions) Dcfh da, by Beyotime (6 mentions)

Close spelling variants of this product

Hoechst 33342 staining solution Antifade Mounting Medium with Hoechst 33342 Hoechst 33342 nuclear blue fluorescent probes Hoechst 33342 (P0133, Bisbenzimide H33342 trihydrochloride (Hoechst 33342) Hoechst 33342 reaction solution Hoechst 33342 fluorescent dye Hoechst 33342 dye solution Hoechst 33342–propidium iodide (PI) double-staining apoptosis-detection kit Hoechst 33342/PI

660 protocols using hoechst 33342

Colony Formation and Apoptosis Assay

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Cells infected with shRNA-KIF21B lentivirus (200 cells/well) were plated in a 6-well plate and cultured for 1-2 weeks. After being xed with 4% paraformaldehyde for 30 min, the cells were stained with 0.1% crystal violet for another 30 min. Finally, we counted the number of colonies.
Hoechst 33342/PI assay Cells infected with shRNAs (1×10 3 cells/well) were cultured in a well plate for 24 h. Then, we co-stained the cells with Hoechst 33342 (10 μg/mL; Beyotime, Shanghai, China) and PI (5 μg/mL; Beyotime) for 15 min. After washing with PBS for 3 times, cell uorescence was observed and captured under a uorescence microscope.

Sun Z., Pan F., Shao J., Yan Q., Lu L., & Zhang N. (2020). Kinesin superfamily protein 21B acts as an oncogene in non-small cell lung cancer.

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Mitochondrial ROS Evaluation in Neurons

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Mitotracker-Red CM-H2X ROS (#M7513, Thermo Fisher, CA) was used to evaluate mitochondrial-derived oxygen-derived free radicals. After 6-h high glucose stimulation, neurons were incubated with Mitotracker-Red CM-H2X ROS solution (1 µM) at 37°C for 15 minutes and Hoechst 33342 (#C1029, Beyotime Biotechnology, Shanghai, China) for 5 minutes.
Brain samples were fixed with 4% paraformaldehyde, blocked in 5% goat serum (Absin Biomart, Shanghai, China) at 37°C for 20 minutes. The samples were incubated with dihydroethidium (10 μM, Beyotime Biotechnology, Shanghai, China) for 1 hour. Hoechst 33342 (#C1029, Beyotime Biotechnology, Shanghai, China) for 5 minutes. Fluorescent signals in hippocampal CA1 region were detected using a fluorescence microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ (NIH, Bethesda, MD).

Hu Y., Zhou Y., Yang Y., Tang H., Si Y., Chen Z., Shi Y, & Fang H. (2022). Metformin Protects Against Diabetes-Induced Cognitive Dysfunction by Inhibiting Mitochondrial Fission Protein DRP1. Frontiers in Pharmacology, 13, 832707.

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Visualizing EV Uptake in Recipient Cells

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To examine the uptake of EVs by recipient cells, the EVs purified from HEK293T cells were stained with the red fluorescent dye PKH26 (Sigma) for 5 min at room temperature, and neutralized using exosome-free FBS. The PKH26-labelled EVs were washed with PBS and centrifuged at 100,000 g for 2 h at 4°C. Huh7 cells were incubated with the resuspended PKH26-labelled EVs for 3 h, followed by washing with PBS. 4% paraformaldehyde (PFA) was used to fix cells at room temperature for 15 min, and Hoechst 33342 (Beyotime) was used to stain the nucleus of cells. The uptake of EVs by Huh7 cells was observed using the TCS-SP8 STED 3X confocal microscope (Leica).
For immunocytofluorescence analysis, cells were fixed in 4% PFA for 15 min and permeabilized with 0.5% TritonX-100 for 15 min, followed by incubation with anti-CD63 antibody (Abcam) overnight at 4°C. Cells were stained with Hoechst 33342 (Beyotime) and observed using the TCS-SP8 STED 3X confocal microscope (Leica).

Zeng W., Zheng L., Li Y., Yang J., Mao T., Zhang J., Liu Y., Ning J., Zhang T., Huang H., Chen X, & Lu F. (2023). Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration. Emerging Microbes & Infections, 13(1), 2284286.

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Hoechst 33342 Staining for Morphological Examination

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Hoechst 33342 staining was done according to our previous study.¹⁶ The morphological changes of nuclei were examined using
membrane-permeable DNA-binding dye Hoechst 33342 (Beyotime). Briefly, the cells
were inoculated in 6-well plate at the concentration of 1 × 10⁵cells/well in 2 mL medium. When reaching 70% to 80% confluence, the cells were
treated with 200, 400, and 600 μg/mL of CTPG or cisplatin for 24 hours. The
cells were washed with PBS and fixed with 4% ice-cold paraformaldehyde at 4°C
for 10 minutes. After washing with PBS, cells were stained with Hoechst 33342 at
4°C for 10 minutes. Samples were observed by fluorescence inverted microscope
(Nikon Eclipse Ti-E, Japan).

Yuan P., Fu C., Yang Y., Adila A., Zhou F., Wei X., Wang W., Lv J., Li Y., Xia L, & Li J. (2021). Cistanche tubulosa Phenylethanoid Glycosides Induce Apoptosis of Hepatocellular Carcinoma Cells by Mitochondria-Dependent and MAPK Pathways and Enhance Antitumor Effect through Combination with Cisplatin. Integrative Cancer Therapies, 20, 15347354211013085.

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Apoptosis Analysis by FCM and Hoechst Staining

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Apoptosis was analyzed using two methods: FCM and Hoechst 33342 staining. In FCM, the cells were collected by trypsinization, washed three times with ice-cold PBS solution, and stained with 500 µL binding buffer containing 5 µL Annexin V-fluorescein isothiocyanate (Beyotime Biotech) and 5 µL PI at room temperature for 15 minutes in the dark; the stained cells were then analyzed by FCM (FC500; Beckman Coulter Inc.). The signals of early and late apoptotic cells were located in the lower and upper right quadrant of the resulting dot plot, respectively. In Hoechst 33342 staining, the cells were washed twice with PBS and stained with Hoechst 33342 (Beyotime Biotech) at 37°C for 20 minutes in the dark. The cells were then washed with PBS and cell nuclei were visualized under the High-Content Imaging System (Perkin-Elmer Operetta).

Zhao X., Qi T., Kong C., Hao M., Wang Y., Li J., Liu B., Gao Y, & Jiang J. (2018). Photothermal exposure of polydopamine-coated branched Au–Ag nanoparticles induces cell cycle arrest, apoptosis, and autophagy in human bladder cancer cells. International Journal of Nanomedicine, 13, 6413-6428.

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Measuring Cell Proliferation with EdU

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Cell proliferation was determined using 5-ethynyl-2’-deoxyuridine (EdU) DNA Cell Proliferation kit (Beyotime, C0071S) following the manufacturer’s protocol. After the required treatment, cells were incubated with 1×EdU working liquid for 2 h and then fixed in 4% PFA for 15 min. After washing, cells are blocked with 0.3%Triton X–PBS for 15 min and then incubated with click reaction liquid for 30 min at room temperature in the dark. The cells were finally mounted using the anti-fade medium containing 1× Hoechst 33342 (Beyotime, C0071S) and observed under an Olympus IX-73 microscope.

Sun T.T., Li X.M., Zhu J.Y., Yao W., Yang T.J., Meng X.R., Yao J, & Jiang Q. (2022). Regulatory effect of long-stranded non-coding RNA-CRNDE on neurodegeneration during retinal ischemia-reperfusion. Heliyon, 8(10), e10994.

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EdU Assay for Cell Proliferation

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Cell proliferation was determined using an EdU assay kit (Beyotime Biotechnology, Shanghai, China). About 1 × 10⁵ cells were incubated in 24-well plates for 24 hours and then treated with si-AC084117.1, si-NC, or nothing for 2 days, followed by incubation with 10 μM EdU at 37°C for 2 hours. The cells were then fixed with 4% paraformaldehyde for 15 minutes, incubated with phosphate-buffered saline containing 0.3% Triton X-100 for 10 minutes, and incubated with Click reaction solution in the dark at room temperature for 30 minutes. Finally, the cells were stained with 1 × Hoechst 33342 (Beyotime Biotechnology, Shanghai, China) for 10 minutes and images were captured using an Olympus BX51 immunofluorescence microscope (Olympus, Tokyo, Japan).

Cai L., Cai L., Zhou L., Zhao Y, & Qian J. (2023). Identification and validation of a seven cuproptosis-associated lncRNA signature to predict the prognosis of endometrial cancer. The Journal of International Medical Research, 51(12), 03000605231213435.

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Quantifying Proliferation in MCF-7R Cells

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MCF-7R cells (5×10⁴ cells/well) were seeded onto 24-well plates and processed with the different drugs (5 mM Met, 1 μM OHT, 20 nM RAPA) for 48 h. The cells were then exposed to 5-ethynyl-2’-deoxyuridine (EdU, RiboBio, China) for 2 h. Next, the cells were fixed with 4% formaldehyde for 30 min and permeabilized with 0.5% Triton-X-100, and then incubated with 100 μL/well of 1× Apollo^® reaction cocktail at room temperature for 30 min. The cells were treated with 1× Hoechst 33342 (Beyotime, China) for 30 min to stain the DNA contents, and finally visualized using a fluorescence microscope.

Jiang Y., Qian T., Li S., Xie Y, & Tao M. (2022). Metformin reverses tamoxifen resistance through the lncRNA GAS5-medicated mTOR pathway in breast cancer. Annals of Translational Medicine, 10(6), 366.

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EdU Incorporation Assay for Proliferation

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Cells were inoculated into 24-well culture plates for 24 h. A concentration of 50 μM 5-ethynyl-2’-deoxyuridine (EDU; C0075S-1, Beyotime, Biotechnology) was added and incubated for 1.5 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 10 min. Subsequently, cells were treated with 200 μL Click Additive Solution (C0075S-5, Beyotime, Biotechnology) at room temperature and protected from light for 30 min. Cells were then stained with 200 μL of 1× Hoechst 33342 (C0075S-6, Beyotime Biotechnology) at room temperature for 30 min under a laser scanning confocal focusing microscope (FV-1000; Olympus, Tokyo, Japan). Five areas of positive cells were randomly counted.

Cheng M., Cao H., Yao P., Guan J., Wu P., Ji H., Jiang S., Yuan Y., Fu L., Zheng Q, & Li Q. (2023). PHF23 promotes NSCLC proliferation, metastasis, and chemoresistance via stabilization of ACTN4 and activation of the ERK pathway. Cell Death & Disease, 14(8), 558.

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Visualizing LvPerilipin Localization in High Five Cells

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The High Five cells were maintained in the Express Five SFM medium (Gibico, New York, USA) with 10% L-Glutamine (Gibico, USA) and were transfected with a pIEX-4 vector or Myc-tagged LvPerilipin expression vector and were cultured for 36 h at 28 °C in 35 mm confocal Petri dishes (Cellvis, Sunnyvale, USA). After washing three times with PBS, 4% paraformaldehyde was used to fix the cells at room temperature for 15 min, and 0.5% TritonX-100 (diluted in PBS) was used to permeabilize the cells for 2 min. After washing, the cells were blocked with QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime, Shanghai, China) for 1 h, followed by incubation overnight (about 10 h) at 4 °C with a mouse anti-Myc antibody (Cell Signaling Technology, USA; 1:300). Then, the cells were subsequently incubated with Alexa Fluor 488 goat anti-mouse (Beyotime, Shanghai, China) for 1 h at room temperature. After washing three times, Nile Red (ThermoFisher Scientific, Waltham, MA, USA) was used to stain the LDs in a final concentration of 0.1 μM for 10 min at room temperature. The cells were washed with PBS five times before the nuclei were stained with 1 × Hoechst 33342 (Beyotime, Shanghai, China) for 10 min. Finally, the cells were observed under a confocal laser-scanning microscope (Carl Zeiss LSM 800, CarlZeiss, Oberkochen, Germany).

Wang C., Yao D., Zhao M., Lu K., Lin Z., Chen X., Zhao Y, & Zhang Y. (2022). Shrimp Lipid Droplet Protein Perilipin Involves in the Pathogenesis of AHPND-Causing Vibrio parahaemolyticus. International Journal of Molecular Sciences, 23(18), 10520.

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