Apoptotic and necrotic cell death were characterized by the use of Hoechst 33342 staining (Molecular Probes). Cells were stained with 0.5 μg/ml Hoechst 33342 for 30 min at 37°C. After washing the cells twice with phosphate buffered saline, the cells were examined under UV light with a digital camera attached to a fluorescence microscope. Apoptotic cells were characterized by fragmented and/or condensed nuclei and necrotic cells by diffuse and irregular nuclei.
Hoechst 33342 staining
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33342 is a fluorescent dye that binds to DNA. It can be used to stain and visualize DNA in various applications, such as flow cytometry and fluorescence microscopy.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
In cell analyzer 2000, by GE Healthcare (2 mentions)
Mouse anti h3cit monoclonal antibody, by BioCat (2 mentions)
In cell investigator software, by GE Healthcare (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (2 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Draq 5 dye, by Biostatus (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Fv3000rs confocal microscope, by Olympus (1 mentions)
Mab1864, by Thermo Fisher Scientific (1 mentions)
A22287, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Leica (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Ultra high resolution laser scanning microscope, by Olympus (1 mentions)
Evos fl auto, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by PanEco (1 mentions)
Antibiotics antimycotics, by Merck Group (1 mentions)
Kga108, by Keygen Biotech (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
21 protocols using hoechst 33342 staining
1
Hoechst 33342 Staining for Cell Death
2
Quantifying Apoptosis in T24 Cells
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Cell apoptosis was assessed using Hoechst 33342 staining (Molecular Probes, Carlsbad, CA, USA). Briefly, replicate cultures of T24 cells were plated in 6-well plates. The cells were treated with or without DEA for 24 hours. After a change of fresh medium, the cells were incubated with Hoechst 33342 solution at 37°C for 10 minutes, followed by examination under a fluorescence microscope. Strong fluorescence and condensed or fragmented nuclei were observed in apoptotic cells, while weak fluorescence was observed in live cells. The quantification of apoptotic cells was performed by taking images in random fields and counting at least 200 cells in four random fields per well. The nuclear DNA in the treated cells contained in 6-well plates was visualized by staining with the DNA-specific dye Hoechst 33342 at a final concentration of 5 μg/ml. The cells were observed immediately with filters for blue fluorescence.
3
High-Content Screening of E-Liquid Toxicity
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NHBE cells were seeded into black, clear-bottom 96-well tissue culture plates at a density of 37,500 cells per cm2 (12,000 cells per well) in 100 μL of culture medium. The cells were incubated for 24 h in the culture medium and then exposed (in three replicate wells) to increasing concentrations of the e-liquid solutions containing the different flavoring substances. A corresponding base solution was included as a reference. The cells were exposed for 30 min (NF-κB endpoint only), 4 h, and 24 h before performing the HCS assays. In parallel, appropriate positive controls at three different concentrations were used for each endpoint (Table S2). Dimethyl sulfoxide (CASRN 67-68-5; purity >99.9%; ref. D8418, Sigma-Aldrich) was used as the vehicle for all positive control treatments at a final concentration of 0.5%. Cell count was recorded in all assays using Hoechst 33342 staining (Life Technologies, Carlsbad, CA, USA), except for the oxidative stress assay, where DRAQ5™ dye (Biostatus, Shepshed, UK) was used. Following staining of the NHBE cells, fluorescence was analyzed by image acquisition with a Thermo Fisher Cellomics® ArrayScan VTI High Content Screening Reader (Thermo Fisher Scientific, Waltham, MA, USA) and vHCS™:View software (Thermo Fisher Scientific). Twenty fields were imaged per well using a 10× wide field objective, as previously described [15 ].
4
Quantification of Histone H3 Citrullination
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Human neutrophils were isolated as above and diluted to a density of 3.5×105/mL. Cells were incubated with compounds in 384-well micro-titre plates for 45 min at 37 °C, 5% CO2. Cells were then stimulated with 2 µM calcium ionophore (final concentration) for 60 min at 37 °C. The cells were fixed with 1.3% PFA for 45 min at RT, then permeabilised in PBS/2% Triton X-100 for 10 min. Cells were washed with PBST (PBS/0.1% Tween 20) and blocked in PBST/2% BSA for 16 h at 4 °C. H3 citrullination was measured using a mouse anti-H3Cit monoclonal antibody (BioCat, 133483, generated at GSK, 6 µg/mL) and a secondary Alexa Fluor 488 goat anti-mouse IgG (Life Technologies, A11001, 2 µg/mL) with parallel Hoechst 33342 staining (Life Technologies). Plates were imaged on an IN Cell Analyzer 2000 (GE Healthcare) and the FITC intensity in neutrophil nuclei was used as a measure of H3 citrullination as determined using the IN Cell Investigator software (GE Healthcare).
5
Quantification of Histone H3 Citrullination
6
Immunohistochemical Analysis of Biopsies
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N(Control)=12 biopsies and N(CTE)=6 biopsies were prepared for immunohistochemical analyses. Biopsies were fixed for 2 h in either 4% formaldehyde or by successive 1 h incubations in cold 70, 90 or 100% methanol solutions and then stored at −20 °C in methanol. The samples were then paraffin embedded. A measure of 5 μm sections were de-waxed in a xylene bath, rehydrated in isopropanol and in solutions with decreasing ethanol concentrations, and were processed for either immunohistochemistry or immunostaining. For immunohistological analyses, classical hematoxylin/eosin staining procedure was used. For immunostaining, de-waxed tissue sections were blocked in 1.5% donkey serum (Sigma-Aldrich, St Louis, Missouri, USA) for 1 h. Primary antibody incubations were performed at 4 °C overnight and secondary antibody incubations at room temperature for 2 h, both in 1.5% donkey serum solution. Hoechst 33342 staining (Life Technologies, Paisley, UK) was used to detect nuclei. Tissue sections were mounted in home-made Mowiol 488 solution.
7
Multiparametric Imaging of SARS-CoV-2 Infection
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WT or D614G-infected LAE ALI cultures were fixed twice for 20 minutes in 4% formaldehyde in PBS and stored in PBS. The SARS-CoV-2 N antigen was stained with polyclonal rabbit anti-SARS-CoV N protein (Invitrogen PA1–41098, 0.5 ug/mL),and using species-specific secondary antibodies as previously described (4 (link)). The cultures were also imaged for a-tubulin (Millipore MAB1864; 3ug/mL) and MUC5AC (Thermo Scientific 45M1; 4ug/mL) as indicated. Filamentous actin was localized with phalloidin (Invitrogen A22287), and nuclei was visualized with Hoechst 33342 staining (Invitrogen). An Olympus FV3000RS confocal microscope in Galvo scan mode was used to acquire 5-channel Z stacks by 2-phase sequential scan. Representative stacks were acquired and are shown as Z-projections and XZ cross sections to distinguish individual cell features and to characterize the infected cell types. ImageJ was used to measure the relative apical culture surface covered by multiciliated cells.
8
Immunofluorescent Imaging of Macrophages and Dendritic Cells
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Sections of paraffin-embedded tumor specimens were deparaffinized, rehydrated in xylene and a series of graded ethanol. Sections were subjected to antigen retrieval with citrate buffer (pH 6.0) and then incubated with fluorophore-conjugated anti-F4/80 (1 :25, RM8, eBioscience) and anti-CD11c (1:50, N418, eBioscience) monoclonal antibodies. Visualization of the nuclei was achieved by Hoechst 33342 staining (1: 20,000, Invitrogen). Sections were mounted with Dako fluorescence mounting medium. Images were taken using a confocal microscope.
9
Immunostaining of Anorectum Tissue
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The recipient’s anorectum (the most distal 1 cm colon) was harvested, fixed in 4% paraformaldehyde overnight and embedded in paraffin. Tissue sections were deparaffinized, rehydrated and then subjected to heat-induced antigen retrieval. After permeabilized with Tween-20 and blocked with 1% bovine serum albumin, the sections were incubated with primary antibodies against GFP (1:200, GeneTex) for 1.5 h, followed by fluorescence-conjugated donkey anti-rabbit IgG (Poly4064, BioLgend), and finally treated with anti-TUBB3 (TUJ1, BioLegend) and anti-GFAP (2E1.E9, BioLegand) directly conjugated with fluorescence. Visualization of the nuclei was achieved by Hoechst 33342 staining (1: 20,000, Invitrogen). Sections were mounted with Dako fluorescence mounting medium. Images were taken using a Leica confocal microscope.
10
Mitochondrial Dynamics in Cardiomyocytes
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Cardiomyocytes were seeded into 48-well plates. After corresponding treatments, cells were washed thrice with PBS and then incubated with 10 nM tetra-methylrhodamine ethyl ester (TMRE) (Invitrogen, United States) for 30 min followed by 10 μg/ml Hoechst 33,42 staining (Solarbio, China) for 10 min at 37°C. Mitochondrial membrane potential and nuclear condensation were detected using EVOS FL Auto (Life Technologies, Bothell, WA, United States).
After washing with PBS, cardiomyocytes were fixed with 4% paraformaldehyde for 15 min and incubated with 0.3% Triton X-100 for 10 min at room temperature; cardiomyocytes were then incubated with 1 μM MitoTracker Red (Invitrogen, United States) for 30 min followed by 10 μg/ml Hoechst 33342 staining for 10 min. Mitochondrial morphology was detected using an ultra-high-resolution laser scanning microscope (Olympus, Japan).
After washing with PBS, cardiomyocytes were fixed with 4% paraformaldehyde for 15 min and incubated with 0.3% Triton X-100 for 10 min at room temperature; cardiomyocytes were then incubated with 1 μM MitoTracker Red (Invitrogen, United States) for 30 min followed by 10 μg/ml Hoechst 33342 staining for 10 min. Mitochondrial morphology was detected using an ultra-high-resolution laser scanning microscope (Olympus, Japan).
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