Double-stranded RNAs (250 ng/well) were pre-spotted in 384-well plates (Ambion AM8500). DL1 cells stably maintaining “pMtnA eGFP MALAT1” were grown for one passage in the absence of hygromycin B. A total of 15,000 cells were then seeded in each well of the 384-well plates in 10 µL of serum-free Schneider's Drosophila media. After 1 h, complete media (with serum) was added (20 µL/well). Cells were grown for 3 d and then treated with 10 µL of media containing CuSO4 (500 µM final concentration, Fisher BioReagents BP346-500) for 6 h. Cells were fixed (5% formaldehyde, Fisher BioReagents BP531-500) and stained with Hoechst 33342 (Sigma B2261) to visualize nuclei. Four images per well (eGFP and Hoechst 33342 staining) were captured at 20× magnification using an automated microscope (ImageXpress Micro, Molecular Devices) and analyzed using MetaXpress software. “Mean stain integrated intensity” of eGFP and “total cell number” were calculated for each image, and the median and interquartile ranges (IQR) were used to calculate a z-score for each plate: (Mean stain integrated intensity-median)/(IQR*74). Wells with robust Z-scores ≥1.3 or ≤ −1.3 were considered hits and Gene Ontology (GO) analysis was performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID), v6.8 (https://david.ncifcrf.gov/home.jsp ) with standard parameters.
Hoechst 33342
Manufactured by Molecular Devices
Sourced in United States
Hoechst 33342 is a fluorescent dye that binds to DNA. It is commonly used in various laboratory applications to stain and visualize nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Merck Group (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Imagexpress micro, by Molecular Devices (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Oxaliplatin, by Merck Group (2 mentions)
Spectramax m2e, by Molecular Devices (2 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (2 mentions)
Am8500, by Thermo Fisher Scientific (1 mentions)
Bp346 500, by Thermo Fisher Scientific (1 mentions)
Suberoylanilide hydroxamic acid saha, by Cayman Chemical (1 mentions)
Suberoylanilide hydroxamic acid, by Cayman Chemical (1 mentions)
Imagexpress micro confocal high content imaging system, by Molecular Devices (1 mentions)
Metaxpress image acquisition and analysis software, by Molecular Devices (1 mentions)
Rabbit anti erbb4, by Cell Signaling Technology (1 mentions)
Imagexpress micro confocal high content microscope, by Molecular Devices (1 mentions)
Digimizer, by MedCalc (1 mentions)
Nile red, by Thermo Fisher Scientific (1 mentions)
Spectramax m2 microplate, by Molecular Devices (1 mentions)
Imagexpress micro confocal system, by Molecular Devices (1 mentions)
Imagexpress ultra, by Molecular Devices (1 mentions)
11 protocols using hoechst 33342
1
High-Throughput RNA Interference Screening
2
Measuring TBP Aggregation Inhibition
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GFP fluorescence was evaluated to reflect TBP aggregation in TBP/Q79-GFP-expressing 293 cells. Briefly, cells were plated on 96-well (2 × 104/well) dishes, grown for 24 h and treated with different concentrations of SG-Tang (0.001−1000 μg/ml), or a positive control suberoylanilide hydroxamic acid (SAHA, 0.1 µM) (Cayman Chemical, Ann Arbor, MI, USA). After 8 h, doxycycline (10 µg/ml) and oxaliplatin (5 µM) (Sigma-Aldrich, St Louis, MO, USA) were added to the cells for 6 days to induce TBP/Q79-GFP expression and inhibit cell cycle progression [71 (link)], respectively. The cells were kept in the medium containing doxycycline, oxaliplatin and SG-Tang for 6 days. On the eighth day, cells were stained with Hoechst 33342 (0.1 µg/ml; Sigma-Aldrich) for 30 min, and images of the cells were automatically obtained using an ImageXpressMICRO high content analysis (HCA) system (Molecular Devices, San Jose, CA, USA) (482 nm excitation and 536 nm emission for enhanced GFP; 377 nm excitation and 447 nm emission for Hoechst 33342). Aggregation was determined by Transfluor technology [72 (link)] based on GFP fluorescence intensity. To quantify aggregation, the relative aggregation level in untreated cells was set as 100%. In addition, IC50 value (half-maximal inhibitory concentration) for SG-Tang was evaluated based on the survived cell number.
3
Evaluating TBP Aggregation Using GFP Fluorescence
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GFP fluorescence was evaluated to reflect TBP aggregation in TBP/Q79-GFP-expressing 293 cells. Briefly, cells were plated on 96-well (2 × 104/well) dishes, grown for 24 h and treated with different concentrations of licochalcone A or LM compounds (0.1 nM−100 μM) or a positive control suberoylanilide hydroxamic acid (SAHA, 100 nM) [46 (link)] (Cayman Chemical, Ann Arbor, MI, USA). After 8 h, doxycycline (10 μg/mL) was added to the cells for 6 days to induce TBP/Q79-GFP expression. In addition, oxaliplatin (5 μM) (Sigma-Aldrich, St Louis, MO, USA) was included for TBP/Q79-GFP aggregate accumulation via blocking cell-cycle progression [47 (link)]. On the eighth day, the cells were stained with Hoechst 33342 (0.1 μg/mL; Sigma-Aldrich) for 30 min, and cell images were automatically recorded (excitation/emission wavelengths of 482/536 nm for GFP and 377/447 nm for Hoechst 33342) by using an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, Sunnyvale, CA, USA). Aggregation was determined by a Transfluor technology [48 (link)] based on GFP fluorescence intensity. To quantify aggregation, the relative aggregation level in untreated cells was set as 100%. Aggregation was measured in wells containing ≥80% viable cells.
4
Tau Aggregation Evaluation Assay
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DsRed fluorescence was evaluated to reflect tau aggregation. On the first day, ∆K280 tauRD-DsRed 293 cells were seeded into the 96-well dish in a density of 0.8 × 104 cells/well and one day after seeding, 5~20 μM Congo red or 50~200 μg/ml Bai-Shao, Gan-Cao, and SG-Tang were added. After 8 h of culture, doxycycline (1 μg/ml; Sigma-Aldrich) was added to induce misfolded tau expression. On the fifth day, cells were stained with Hoechst 33342 (0.1 μg/ml) for 30 min, and fluorescence intensities (543 nm excitation and 593 nm emission for DsRed; 377 nm excitation and 447 nm emission for Hoechst 33342) were measured using a high content analysis (HCA) system (ImageXpressMICRO, Molecular Devices). All images were analyzed by MetaXpress Image Acquisition and Analysis Software (Molecular Devices).
5
Multicolor Immunofluorescence Microscopy
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For immunofluorescence microscopy, 2500 cells per well were seeded in a flat‐bottom 384‐well plate and allowed to attach overnight. Cells were then fixed in 4% PFA, permeabilized in 0.1% Triton/PBS and blocked in 3% BSA + 0.05% Triton in PBS. The primary antibody rabbit anti‐ERBB4 (#4795, Cell Signaling Technology) was diluted 1 : 100, and the primary antibody mouse anti‐P‐glycoprotein (#Ab00143‐1.1, Absolut Antibody Ltd, Redcar, UK) was diluted 1 : 250. Secondary antibodies donkey anti‐rabbit IgG labeled with Alexa Fluor 508 (#A10042, ThermoFisher Scientific Inc.) and goat anti‐mouse IgG labeled with Alexa Fluor 488 (#4408S, Molecular Probes, ThermoFisher Scientific Inc.) were diluted 1 : 500 and 1 : 1000, respectively, and stained concurrently with 0.5 μg·mL−1 Hoechst 33342 (#H3570, ThermoFisher Scientific Inc.). Between steps, the cells were washed three times with PBS. Images were taken using an ImageXpress Micro Confocal high content microscope (Molecular Devices, San Jose, CA, USA) using Cy3 (ERBB4), FITC (P‐gp), DAPI (Hoechst 33342), and brightfield channels.
6
Modulation of 3T3-L1 Adipogenesis by Calcium Channel Blockers
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TTA-A2, IC50 ~5 µM (Kraus et al. 2010 (link)) and Calciseptine IC50 ~10 nM (De Weille et al. 1991 (link)) selective blockers of CaV3.x and CaV1.x, respectively, as well as mibefradil, which has 12–13 greater potency on CaV3.x, IC50 ~1 µM than CaV1.x (Martin et al. 2000 (link)) (Alomone, Jerusalem, Israel), were applied to 3T3-L1 cells prior to and post differentiation with each media exchange. On the eighth day, cell proliferation was determined by nuclear density and differentiation by lipid content. Nuclei were stained with Hoechst 33342 (ThermoFisher) at 10 µg mL-1 in Hanks’ for 30 min. After twice washing in PBS, lipid was stained by 10 µg mL−1 Nile Red (NR) (ThermoFisher) in Hanks’ for 10 min. Dye content was detected with a SpectraMax M2 microplate spectrophotometer (Molecular Devices) with 350 nm excitation/490 nm emission for Hoechst 33342 and 510 nm excitation/590 nm emission for NR. After background correction, the NR to Hoechst fluorescence ratio was taken as an index of adipogenesis. Images were captured with a Zeiss ERc 5rs Axiocam attached to an Zeiss Axio with a 20× objective and filter sets as indicated earlier. Images were acquired with Zeiss Zen software and analysed with Digimizer (MedCalc Software Ltd).
7
Nuclear Staining with Hoechst 33342
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The nucleic acid stain Hoechst 33342 (Sigma-Aldrich) was used as a cell-permeant nuclear counterstain, as described previously (40 (link)). Briefly, cells were washed with PBS and then incubated with 3 μM Hoechst 33342 for 15 min at 37 °C with 5% CO2; fluorescence visualization was confirmed with an ImageXpress Micro Confocal system (Molecular Devices).
8
Quantifying Focus Formation in ARPE-19 Cells
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ARPE-19 cells were cultured in 96-well plates for 5 days and then exposed for 2 days to TNF-α (100 ng/ml, eBioscience) and TGF-β2 (5 ng/ml, eBioscience) in the absence or presence of SB431542 (10 µM, Calbiochem) or test drugs. They were then fixed for 30 min at room temperature with 4% paraformaldehyde in PBS, washed with PBS, incubated for 60 min at room temperature with Hoechst 33342 (Invitrogen) and phalloidin–Alexa Fluor 568 (Invitrogen) in PBS, and washed again with PBS. The cells were examined with a high-throughput image screening system (ImageXpress ULTRA, Molecular Devices) for quantitation of focus formation, with a focus being defined as a cell aggregate with an area of Hoechst 33342 fluorescence greater than a certain threshold. The fluorescence intensity of each focus was measured, and the percentage inhibition of focus formation by test drugs was determined as: 100 – [100 × (fluorescence intensity for all foci in the presence of the test drug)/(fluorescence intensity for all foci in the absence of the drug)]. The median inhibitory concentration for each drug was determined from the concentration-response curve.
9
Apoptosis Analysis of Irradiated Cells
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SAS cells or HSC‐2 cells were exposed to 6 Gy irradiation and then treated with EVs or transfected with miRNA mimic and siRNA. After 48 h, cells were stained with Apotracker Green (BioLegend, San Diego, CA, USA) and Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA). The fluorescence intensity ratio (Apotracker Green: 500 nm (Ex), 520 nm (Em); Hoechst 33342: 350 nm (Ex), 461 nm (Em)) were calculated using a fluorescence plate reader (SpectraMax i3x; Molecular Devices, San Jose, CA, USA).
10
Mitochondrial ROS Measurement Across Neurological Models
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