Hoechst 33342

Manufactured by Dojindo Laboratories

Sourced in Japan, United States, China

Hoechst 33342 is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in various biological applications for the detection and analysis of cell nuclei.

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Hoechst 33342 staining Cellstain Hoechst 33342 solution Hoechst 33342 solution Hoechst 33342 dye Hoechst 33342 stain Hoechst 33342 (H342, Hoechst

297 protocols using hoechst 33342

Lanthanide Ion-Based Multicolor Staining

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After rinsing with HEPES buffer solution (10 mM HEPES, pH = 7 adjusted with NaOH), immersion in 95 or 99.5% ethanol, and rinsing with the HEPES buffer, cells and tissues were immersed in 100% D₂O HEPES buffer solution containing TbCl₃ (TBH03XB, Kojundo Chemical Laboratory) at concentrations of 0.1–100 mM for 1–5 min. The cells and tissues were rinsed with 100% D₂O HEPES buffer solution. For multicolor staining, a 100% D₂O HEPES buffer solution containing TbCl₃ and Hoechst 33342 (Dojindo Molecular Technologies) or DAPI (Dojindo Molecular Technologies) at the concentrations of 0.1–100 mM and 10–20 µg/ml, respectively, were used. For temperature-dependent staining experiments, the staining solution containing Tb³⁺ at the concentration of 50 mM was heated to 50, 60, 70, 80, and 90 °C prior to application to the ethanol-treated cells, and the cells were subsequently immersed in the heated solution for 5 min at target temperature.
For the conventional staining procedures using Rhodamine B (Nacalai esque), eosin Y (Merck), PI (Dojindo Molecular Technologies), and Hoechst 33342, a buffer solution containing one of those dyes at the concentration of 100 µg/ml (for Rhodamine and PI), 1 mM (eosin Y), or 2 µg/ml was used. For tissue staining, the concentrations of Hoechst 33342 and Rhodamine B were adjusted to 20 and 100 µg/ml, respectively.

Kumamoto Y., Matsumoto T., Tanaka H, & Takamatsu T. (2019). Terbium ion as RNA tag for slide-free pathology with deep-ultraviolet excitation fluorescence. Scientific Reports, 9, 10745.

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MitoPY1 Staining of Ciliated Cells

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MitoPY1 staining was performed according to Dickinson et al.¹⁰⁵ (link) Briefly, SH-SY5Y cells (RD3 wildtype and knockout cells) were seeded onto a 6-well culture plate (Nunc) at 1×10⁵ cells per well, incubated at 37°C, 5% CO₂ for 24 h, and ciliogenesis was induced. After 24 h ciliogenesis induction, cells were detached with 150 μL Accutase (Nacalai), mixed with 850 μL DPBS, centrifuged at 4°C, 400 G for 5 min, resuspended with 250 μL DPBS containing 10 μM MitoPY1 (Tocris) and 2 μg/mL Hoechst 33342 (Dojindo), incubated at 37°C for 60 min, centrifuged at 4°C, 400G for 5 min, resuspended with 200 μL DPBS, then analyzed by Synergy H1 plate reader (MitoPY1: ex.488 nm/em.527 nm; Hoechst33342: ex.350 nm/em.461 nm). The raw MitoPY1 signal was normalized by Hoechst33342 signal, and multiplied by 1×10⁴ for quantification.

Noguchi Y., Onodera Y., Miyamoto T., Maruoka M., Kosako H, & Suzuki J. (2024). In vivo CRISPR screening directly targeting testicular cells. Cell Genomics, 4(3), 100510.

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Multimodal Cell Viability Assay

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Propidium iodide (PI, Dojindo, Japan), calcein-acetoxymethyl (calcein-AM, Dojindo) and Hoechst 33342 (Dojindo) were incubated with the cells at 37 °C and 5% CO₂ for 15 min at a final concentration of 1, 1.5, and 3.6 μM, respectively, and then were washed once for Inverted Fluorescence Microscope analysis or photographed by a high-throughput cell imaging system at the following fluorescent wavelengths: PI, λex = 530 nm and λem = 580 nm; calcein-AM, λex = 490 nm and λem = 515 nm; and Hoechst33342, λex = 350 nm and λem = 460 nm. Trypan Blue (Sigma, USA) staining was performed at a final concentration of 0.01% in cell medium and then observed under optical light using an inverted microscope.

Zhang Y., He W., He C., Wan J., Lin X., Zheng X., Li L., Li X., Yang X., Yu B., Xian X., Zhu Y., Wang Y., Liu G, & Lu N. (2019). Large triglyceride-rich lipoproteins in hypertriglyceridemia are associated with the severity of acute pancreatitis in experimental mice. Cell Death & Disease, 10(10), 728.

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Intracellular and Mitochondrial Calcium Visualization

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The detection of calcium distribution was visualized by staining with Fluo4-AM (37 °C for 30 min; Dojindo, Japan) and Rhod2-AM (4 °C for 30 min; Dojindo, Japan) for the intracellular and intramitochondrial calcium, respectively. The nuclei were visualized by staining with Hoechst 33342 (Dojindo, Japan).

Zhu Y., Gu H., Yang J., Li A., Hou L., Zhou M, & Jiang X. (2024). An Injectable silk-based hydrogel as a novel biomineralization seedbed for critical-sized bone defect regeneration. Bioactive Materials, 35, 274-290.

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Quantifying Cholesterol Uptake in Bovine Sperm

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BODIPY-cholesterol (TopFluor^® Cholesterol 810255, Nanocs, NY, USA) was used for the quantification of the incorporated cholesterol. BODIPY-labeled cholesterol was dissolved by dimethyl sulfoxide at 50 mg/ml, and it was used as the stock solution. Just after thawing of the frozen-thawed bovine sperm, the sperm were washed with base medium containing BODIPY-cholesterol at different concentrations. After centrifugation for 5 min (500g, 37°C) and washing twice with base medium to remove excess BODIPY-cholesterol, trypan blue staining (0.4%) (355-25, Nakalai Chemicals Ltd, Kyoto, Japan) was performed to quench fluorescence signals on the surface of the sperm to detect the actual region of cholesterol uptake. The fluorescence level was analyzed with an Attune^® NxT Acoustic Focusing Cytometer using a 488 nm laser and a filter with a bandwidth of 530/30 nm, and the intensity of the average value was measured. The sperm population is shown in Supplementary Fig. S1D. Digital images were captured using a Nikon confocal microscope after nucleic visualization by Hoechst33342 (346-0795, Dojindo, Kumamoto, Japan).

Islam M.M., Umehara T., Tsujita N., Koyago M, & Shimada M. (2023). Treatment with cholesterol just after thawing maintains the fertility of bull sperm. Molecular Human Reproduction, 29(9), gaad031.

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Immunofluorescence Staining of Avian PGCs

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Circulating PGCs derived from HH14 embryonic blood or cultured PGCs were adhered onto a MAS-GP Type A coated
glass slide (Matsunami Glass, Osaka, Japan) and fixed with 4% paraformaldehyde for 5 min at room temperature
(RT). After several washes, cells were blocked with PBS containing 5% normal goat serum or Image-iT signal
enhancer (Life Technologies) and incubated overnight at 4ºC with primary antibodies. Then, cells were
incubated for 30 min or 1 h at RT with secondary antibodies. Subsequently, cells were counterstained with 1
µg/ml Hoechst 33342 (Dojindo, Kumamoto, Japan). Fluorescent images were captured using an Eclipse E1000
fluorescence upright microscope (Nikon, Tokyo, Japan), and these images were processed using Photoshop
Elements (Adobe Systems, San Jose, CA, USA) for trimming and overlaying. Sources and dilution of used
antibodies were as follows: rat anti-chicken vasa homolog (CVH) raised in our laboratory (1:10000) [27 (link)], mouse anti-stage specific embryonic antigen-1 (SSEA-1; 1:100,
Developmental Studies Hybridoma Bank, Iowa City, IA, USA), mouse anti-chicken c-KIT (1:500, SouthernBiotech,
Birmingham, AL, USA), goat anti-rat IgG conjugated with Alexa Fluor 488 (1:1000, Life Technologies) and goat
anti-mouse IgG or IgM conjugated with Alexa Fluor 594 (1:1000, Life Technologies).

MIYAHARA D., OISHI I., MAKINO R., KURUMISAWA N., NAKAYA R., ONO T., KAGAMI H, & TAGAMI T. (2015). Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2. The Journal of Reproduction and Development, 62(2), 143-149.

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Desiccation-Rehydration of Pv11 Cells

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Pv11 cells were subjected to desiccation-rehydration (Figs. 1d, 2d, 4, Fig. S4d) as described previously^{3 (link)}. Briefly, cells were incubated in preconditioning medium (600 mM trehalose containing 10% (v/v) complete IPL-41 medium) for 48 h at 25 °C, and then suspended in 400 µL preconditioning medium. Forty microliter aliquots of the cell suspension were dropped into 35-mm petri dishes, and the dishes were desiccated and maintained at < 10% relative humidity and 25 °C for more than 10 days. An hour after rehydration by complete IPL-41 medium, cells were stained with propidium iodide (PI; Dojindo) for dead-cell detection and Hoechst 33342 (Dojindo) for whole-cell detection, and images were acquired using a conventional fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). The survival rate was calculated as the ratio of the number of live cells (Hoechst-positive and PI-negative) to that of total cells (Hoechst-positive).

Miyata Y., Fuse H., Tokumoto S., Hiki Y., Deviatiiarov R., Yoshida Y., Yamada T.G., Cornette R., Gusev O., Shagimardanova E., Funahashi A, & Kikawada T. (2021). Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells. Scientific Reports, 11, 19698.

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Immunofluorescence Characterization of hiPSC-Derived Cardiomyocytes

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hiPSC-derived cardiomyocytes were fixed with 4% PFA and permeabilized with 0.1% Triton-X in PBS (-) for 15 min at 4°C. Then, the cells were blocked with 5% BSA in PBS (-) for 60 min at room temperature. Primary antibodies were reacted for 24 h at 4°C, and secondary antibodies were reacted for 1 h at room temperature. Nuclei were labeled with Hoechst 33342 (Dojindo, H342). Primary antibodies were anti-Troponin T (clone 13-11) (1:200, Thermo Scientific, MA5-12960), anti-MLC2a (1:200, Synaptic Systems, 311 011), and anti-MLC2v (1:200, ProteinTech, 10906-1-AP). Secondary antibodies were Alexa Fluor 488, donkey anti-mouse IgG (HCL), Alexa Fluor 568, donkey anti-rabbit IgG (HCL), Alexa Fluor 647, donkey anti-mouse IgG (HCL). Fluorescence images were obtained using Operetta high content imaging system (PerkinElmer, Japan) and analyzed using Harmony analysis software (PerkinElmer, Japan).

Honda Y., Li J., Hino A., Tsujimoto S, & Lee J.K. (2021). High-Throughput Drug Screening System Based on Human Induced Pluripotent Stem Cell-Derived Atrial Myocytes ∼ A Novel Platform to Detect Cardiac Toxicity for Atrial Arrhythmias. Frontiers in Pharmacology, 12, 680618.

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Internalization of Fluorescent Microvesicles in Cell Lines

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RAW264.7, CMT-93, and Caco-2 cells were plated on a cover glass placed in a 24-well plate. In the case of CMT-93 and Caco-2, cells were starved in serum-free medium for 2 h before the addition of MVs. Cells were then treated with 50 or 100 μg/ml FITC-labeled MVs and incubated at 37°C for 1 h. After the incubation, cells were washed with PBS three times. Regarding immunofluorescence, cells were fixed in 3.7% (w/v) formaldehyde in PBS for 15 min and permeabilized with 0.1% (v/v) Triton X-100 in 1% (w/v) BSA-containing PBS for 15 min. Cells were then treated with Alexa Fluor 568-conjugated phalloidin (Thermo Fisher Scientific) and Hoechst 33342 (DOJINDO). After washing with PBS, cells were observed/photographed using the laser scan confocal microscope IX71 (PLYMPUS). To quantify internalized MVs, cells were lysed with passive lysis buffer (Promega). Hoechst 33342 (final 1/2,000) was added to the cell lysate, and fluorescence intensity was measured with the plate reader Spark (Tecan).

Kobayashi N., Abe K., Akagi S., Kitamura M., Shiraishi Y., Yamaguchi A., Yutani M., Amatsu S., Matsumura T., Nomura N., Ozaki N., Obana N, & Fujinaga Y. (2022). Membrane Vesicles Derived From Clostridium botulinum and Related Clostridial Species Induce Innate Immune Responses via MyD88/TRIF Signaling in vitro. Frontiers in Microbiology, 13, 720308.

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Monitoring Protein-Protein Interactions with Fluoppi

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The Fluoppi system was used to monitor protein-protein interactions in cells. HEK293T cells were transfected as described above. At 16 h after co-transfection, the cells were stained with Hoechst33342 (Dojindo) for 30 min. Image acquisition was performed using IN Cell Analyzer 6000 (GE Healthcare) with a 20× objective lens. Azami-Green (AG) and blue (Hoechst33342) fluorescence on the images was used to count the fluorescent puncta of complexes and nuclei, respectively. Five images were obtained per well. The numbers of fluorescent puncta and nuclei in the five images were counted using ImageJ software [25 (link),30 (link)]. Each image contained approximately 500 cells. The number of fluorescent foci was calculated as the average value for one of the five images, which was normalized to 500 nuclei. The standard errors of the number of fluorescent puncta were calculated from the normalized number of fluorescent puncta.

Omagari K., Asamitsu K, & Tanaka Y. (2021). Application of fluorescent-based technology detecting protein-protein interactions to monitor the binding of hepatitis B virus X protein to DNA-damage-binding protein 1. Biophysics and Physicobiology, 18, 67-77.

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