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Hoescht 33342 dye

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Hoescht 33342 dye is a fluorescent stain that binds to DNA. It can be used to visualize and quantify DNA in various applications, such as flow cytometry and fluorescence microscopy.

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Lab products found in correlation

Esirna library, by Merck Group (2 mentions) Insight nxt high content microscopy system, by Thermo Fisher Scientific (2 mentions) Ab5622, by Merck Group (1 mentions) Aldefluor kit, by STEMCELL (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Eclipse 50i epifluorescence microscope, by Nikon (1 mentions) Phenol red free dmem f12 medium, by Thermo Fisher Scientific (1 mentions) Cellvoyager 7000s, by Yokogawa (1 mentions)

Spelling variants of this product

Hoescht 33342 (142 citations) Hoescht (48 citations) Hoescht dye (8 citations)

5 protocols using hoescht 33342 dye

1

Dendritic Complexity Quantification

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Cultured Hippocampal neurons were stained for MAP2 (1:1000, AB5622 Merck Millipore) and Tau-1 (1:1000 MAB3420, Merk millipore) to distinguish dendrites and axons, respectively, and counterstained with Hoescht 33342 dye (Thermo Fisher Scientific). z-stack images were obtained using Zeiss AxioObserver Z1 and later processed using SNT plugins on FIJI to obtain a traced 2D binary representation of the dendrites for Sholl analysis using 10 μm steps and the number of crossings were counted until a radius of 150 μm. Genotypes were compared using Mixed-Effects analysis. We also evaluated the total length and axonal length and genotypes were compared using KS tests.
Tillotson R., Yan K., Ruston J., DeYoung T., Córdova A., Turcotte-Cardin V., Yee Y., Taylor C., Visuvanathan S., Babbs C., Ivakine E.A., Sled J.G., Nieman B.J., Picketts D.J, & Justice M.J. (2023). A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects. Human Molecular Genetics, 32(15), 2485-2501.
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2

High-Content Screening of Osteosarcoma

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We generated a completely different HCS than the tauopathy study in order to assess the ML method’s generalizability. This osteosarcoma dataset was subjected to different biological conditions. U2OS cells (female) expressing a stable CCNB1-GFP construct were plated into 384-well plates with 500 cells per well and reverse transfected with an esiRNA library (10 ng, Sigma Aldrich) using Hiperfect transfection reagent. The U2OS cells were cultured in DMEM medium containing 10% foetal bovine serum. The library can be found at https://iccb.med.harvard.edu/sigma-esirna-human-1 and also https://iccb.med.harvard.edu/sigma-esirna-human-2. Our method was inspired by the assay presented in Bray et al.57 (link) We performed 16,194 unique functional genomic perturbations. After 72 hours, cells were stained with 5 μg/mL of Hoescht 33342 dye (ThermoFisher), incubated for 60 minutes at 37°C, washed with PBS, fixed in 4% PFA, and scanned on a Thermo Cell Insight NXT high content microscopy system using a 10X objective. We captured the two channels, resulting in 324,989 Hoechst and cyclin-B1 image pairs, each of dimension 1104×1104 pixels.
Wong D.R., Conrad J., Johnson N., Ayers J., Laeremans A., Lee J.C., Lee J., Prusiner S.B., Bandyopadhyay S., Butte A.J., Paras N.A, & Keiser M.J. (2022). Trans-channel fluorescence learning improves high-content screening for Alzheimer’s disease therapeutics. Nature machine intelligence, 4(6), 583-595.
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3

ALDH-CD44+ Cancer Stem Cell Analysis

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MKN45 (5000) cells were plated in non-adherent 6-well plates and 5-day-old spheres were treated or not for 48 h with LIF before harvesting. ALDEFLUOR Kit (STEMCELL Technologies, Grenoble, France) was used to detect ALDH activity, according to the manufacturer’s instructions, prior to spheres incubation with 1:25 anti-human CD44-PE antibody (515 clone, BD Biosciences, Le Pont de Claix, France) in ice-cold buffer containing PBS-0.5% bovine serum albumin (BSA, Gibco, ThermoFisher Scientific, Villebon sur Yvette, France)-2 mM EDTA (Sigma-Aldrich, Saint-Quentin Fallavier, France) for 25 min at 4 °C. Spheres were rinsed twice with ice-cold buffer and incubated for 30 min at RT with Hoescht-33342 dye (ThermoFisher Scientific, Villebon sur Yvette, France). Live immunofluorescence acquisition was carried out with an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) using the NIS-BR acquisition software and a ×40 objective (numerical aperture, 1.3) immediately after buffer-washing and slide mounting. CSC subcellular populations were analysed using the ImageJ 1.52p software (National Institutes of Health) [44 (link)].
Seeneevassen L., Giraud J., Molina-Castro S., Sifré E., Tiffon C., Beauvoit C., Staedel C., Mégraud F., Lehours P., Martin O.C., Boeuf H., Dubus P, & Varon C. (2020). Leukaemia Inhibitory Factor (LIF) Inhibits Cancer Stem Cells Tumorigenic Properties through Hippo Kinases Activation in Gastric Cancer. Cancers, 12(8), 2011.
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4

Peptide-Induced Cell Imaging Assay

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Prior to assay, cells were treated with candidate peptides at concentration of 10 µM. Control was treated with DMSO. Cells were incubated with peptides for 1 h at 37 °C. This was followed by 2 washes and exchange to DMEM/F12 Phenol Red-free Medium (Thermo Fischer Scientific) containing Hoescht 33342 dye (#62249, Thermo Fischer Scientific). After 10 min samples were live imaged on Cell Voyager 7000S or 8000 microscope (Yokogawa) at 37 °C with 40 × oil or 20 × and 60 × water objectives applying the same imaging settings to all the conditions (Exposure time 250 ms, Laser power 30%, Binning 2 × 2 for all the channels). For the image visualization, the same brightness/intensity settings were used for all the samples within the same experiment.
Ivanova A., Kohl F., González-King Garibotti H., Chalupska R., Cvjetkovic A., Firth M., Jennbacken K., Martinsson S., Silva A.M., Viken I., Wang Q.D., Wiseman J, & Dekker N. (2024). In vivo phage display identifies novel peptides for cardiac targeting. Scientific Reports, 14, 12177.
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5

High-Content Screening of Osteosarcoma

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We generated a completely different HCS than the tauopathy study in order to assess the ML method’s generalizability. This osteosarcoma dataset was subjected to different biological conditions. U2OS cells (female) expressing a stable CCNB1-GFP construct were plated into 384-well plates with 500 cells per well and reverse transfected with an esiRNA library (10 ng, Sigma Aldrich) using Hiperfect transfection reagent. The U2OS cells were cultured in DMEM medium containing 10% foetal bovine serum. The library can be found at https://iccb.med.harvard.edu/sigma-esirna-human-1 and also https://iccb.med.harvard.edu/sigma-esirna-human-2. Our method was inspired by the assay presented in Bray et al.57 (link) We performed 16,194 unique functional genomic perturbations. After 72 hours, cells were stained with 5 μg/mL of Hoescht 33342 dye (ThermoFisher), incubated for 60 minutes at 37°C, washed with PBS, fixed in 4% PFA, and scanned on a Thermo Cell Insight NXT high content microscopy system using a 10X objective. We captured the two channels, resulting in 324,989 Hoechst and cyclin-B1 image pairs, each of dimension 1104×1104 pixels.
Wong D.R., Conrad J., Johnson N., Ayers J., Laeremans A., Lee J.C., Lee J., Prusiner S.B., Bandyopadhyay S., Butte A.J., Paras N.A, & Keiser M.J. (2022). Trans-channel fluorescence learning improves high-content screening for Alzheimer’s disease therapeutics. Nature machine intelligence, 4(6), 583-595.
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