Cell apoptotic rates were determined using the Annexin V Apoptosis Detection Kit PE or FITC according to the manufacturer's instructions (#88810272 and 88800572; Invitrogen, Carlsbad, CA, USA). Briefly, cells were harvested, washed with PBS and resuspended in 1× Binding Buffer to 1 × 106 cells/mL. 5 μL Annexin‐V‐PE (or Annexin‐V‐FITC) and 5‐μL PI were added to 100 μL of the cell suspension and incubated for 15 minutes in the dark at room temperature. After incubation, 400 μL 1× Binding Buffer was added. Cells were analyzed using a flow cytometer. For Hoechst 33342 staining, cells (2 × 105/mL) were harvested and added to 10 μL Hoechst 33342 (Keygen Biotech, Nanjing, China) for 15 minutes in the dark at 37°C. After centrifuging for 5 minutes and washing with PBS, 1 mL of Buffer A solution (Keygen Biotech, Nanjing, China) was added at room temperature. Finally, 20 μL of cell pellet was dropped onto a glass slide, and the cells were observed using the fluorescence microscope.
Hoechst 33342
Manufactured by Keygen Biotech
Sourced in China, Germany
Hoechst 33342 is a fluorescent dye used for labeling and visualizing nucleic acids, particularly DNA, in biological samples. It binds to the minor groove of DNA and emits blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Inverted fluorescence microscope, by Leica (2 mentions)
Lysotracker red, by Keygen Biotech (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Salvianolic acid b, by Yuanye Bio-Technology (1 mentions)
Anti cyclinb1, by Boster Bio (1 mentions)
Cck 8, by Beyotime (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Potassium permanganate kmno4, by Sinopharm (1 mentions)
L lactide, by Merck Group (1 mentions)
ε caprolactone, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Chlorin e6 ce6, by Frontier Scientific (1 mentions)
Reactive oxygen species assay kit, by Keygen Biotech (1 mentions)
Dp71 fluorescence microscope, by Olympus (1 mentions)
Biosciences facscan 2 cytometer, by BD (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Keygen Biotech (1 mentions)
35 protocols using hoechst 33342
1
Apoptosis Detection using Flow Cytometry and Fluorescence Microscopy
2
Apoptosis Assay for IBV Infection
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HD11 cells were pre-incubated in 96-well plates and infected with IBV at 10 MOI. To assess apoptosis, the condensed and fragmented nuclei were observed using Hoechst 33342 staining (KeyGEN Biotech, Nanjing, Jiangsu Province, China). At the specified time points, the cells were immobilized with 4% paraformaldehyde (KeyGEN Biotech, Nanjing, Jiangsu Province, China) for 30 min and then incubated with Hoechst 33342 (KeyGEN Biotech) in the dark for 15 min. The typical apoptotic morphological changes were observed using a fluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan) with UV excitation at 350 nm.
3
Cellular Uptake and Trafficking of siRNA
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For microscopic observation on cellular uptake of siRNA, FAM-siRNA was used as donor for green fluorescence. Hoechst 33,342 (KeyGen BioTECH, Nanjing, Jiangsu, China) and DiI (KeyGen BioTECH, Nanjing, Jiangsu, China) were used to stain the nuclei and the membrane of cells and as donors for blue and red fluorescence, respectively. Cells at 6 hours post-transfection (transfected by either Ca-PS lipopolyplex or Lipo2000) were imaged by a Zeiss Axio Vert.A1microscope equipped with an Olympus X-cite 120 Q light source.
For microscopic observation on intracellular trafficking of siRNA, FAM-siRNA was used as donor for green fluorescence. Hoechst 33,342 and LysoTracker-Red (KeyGen BioTECH, Nanjing, Jiangsu, China) were used to stain the nuclei and lysosomes of BMDMs and as donors for blue and red fluorescence, respectively. BMDMs at 1, 6, 12, 24 hours post-transfection (transfected by either Ca-PS lipopolyplex or Lipo2000) were imaged by a Zeiss Axio Vert.A1microscope equipped with an Olympus X-cite 120 Q light source.
For microscopic observation on intracellular trafficking of siRNA, FAM-siRNA was used as donor for green fluorescence. Hoechst 33,342 and LysoTracker-Red (KeyGen BioTECH, Nanjing, Jiangsu, China) were used to stain the nuclei and lysosomes of BMDMs and as donors for blue and red fluorescence, respectively. BMDMs at 1, 6, 12, 24 hours post-transfection (transfected by either Ca-PS lipopolyplex or Lipo2000) were imaged by a Zeiss Axio Vert.A1microscope equipped with an Olympus X-cite 120 Q light source.
4
Oligodendrocyte Death Assay with Nogo-A
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Oligodendrocytes with or without up-/down-regulated Nogo-A were incubated in culture medium containing 2 μg/mL propidium iodide (PI) (Cat# KGA215; Jiangsu Keygen Biotech) and 2 μg/mL Hoechst 33342 (Cat# KGA215; Jiangsu Keygen Biotech) at 37°C for 15 minutes. The percentage of cell death was calculated by PI (+)/Hoechst (+) observed with a fluorescence microscope. Cell death rates were also calculated when Ad-ZsGreen-Nogo-A or Ad-shRNA-Nogo-A was added to the oligodendrocyte cultures 2 hours before, simultaneously with, or 30 minutes after H2O2 treatment.
5
Medicinal Compound Extraction from S. miltiorrhiza
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S. miltiorrhiza medicinal slices were purchased from the Anhui Renpu Pharmaceutical Co. (Anhui, China). The AB-8 macroporous adsorption resin (lot no. 312L011) was obtained from Beijing Solebo Technology Co. (Beijing, China). SAA, salvianolic acid B, and OSR (purity ≥ 98.0%) were purchased from Shanghai Yuanye Biotechnology (Shanghai, China). CCK8 was purchased from Beyotime (Shanghai, China). Hoechst 33342, propidium iodide (PI), and RNase were purchased from Keygen Biotech (Shanghai, China). Anti-CyclinB1 and anti-CDK1 antibodies were purchased from Boster Biotechnology (Shanghai, China). Analytical grade hydrochloric acid and other chemicals were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China).
6
Multifunctional Nanoparticle-Mediated Photodynamic Therapy
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Potassium permanganate (KMnO4) was purchased from Sinopharm Chemical Reagent Co., Ltd. Photosensitizer chlorin e6 (Ce6) was obtained from Frontier Scientific (Logan, UT, USA). Poly (allylamine hydrochloride) (PAH, Mw ~17,500), poly (ethylene glycol) (PEG, Mn 4000), D, L-lactide (LA), ε-caprolactone (ε-CL, Mn 114), stannous octoate (Sn(Oct)2), poly (vinyl alcohol) (PVA, 87-90%, hydrolyzed, Mw 30,000-70,000) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification. Doxorubicin hydrochloride (DOX) was commercially available from Meilune Biological Co., Ltd (Dalian, China). Dulbecco's modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL (Carlsbad, CA, USA). Milli-Q water (18.2 MΩ cm) from a Milli-Q Gradient System was used throughout the experiments. PEG was vacuum dried at 60 °C for 12 h before use, and other materials used in this article were of analytical grade. Hoechst 33342, Reactive Oxygen Species (ROS) Assay Kit and Annexin V-FITC Apoptosis Detection Kit were purchased from KeyGEN BioTECH Co., Ltd (Nanjing, China).
7
Quantifying Mitochondrial Oxidative Stress
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Mitochondrial ROS production was measured using Mito-SOX™ Red mitochondrial superoxide indicator (Invitrogen). Cells were seeded on coverslips and washed with PBS, and then pre-incubated with 1 μM Mito-SOX Red in PBS for 20 min at 37 °C in the dark. After treatments, Hoechst 33342 (KeyGEN BioTECH, China) was added for 20 min. The coverslips were mounted on slides for examination. Images were taken using an Olympus DP71 fluorescence microscope.
For flow cytometer analysis of intracellular ROS levels, cells were washed and harvested in PBS, and then separately stained with 1 μM Mito-SOX Red, 5 μM dihydroethidium (DHE), 10 μM DCFH-DA for 20 min at 37 °C and 5% CO2 in the dark. Samples were subsequently washed using ice-cold PBS and centrifuged for 5 min at 1500 rpm before being resuspended in ice-cold PBS and kept on ice until analysis. Flow cytometry was performed using a BD Biosciences FACScan II cytometer (Becton Dickinson, San Jose, CA, USA). At least 10,000 cells were collected.
For flow cytometer analysis of intracellular ROS levels, cells were washed and harvested in PBS, and then separately stained with 1 μM Mito-SOX Red, 5 μM dihydroethidium (DHE), 10 μM DCFH-DA for 20 min at 37 °C and 5% CO2 in the dark. Samples were subsequently washed using ice-cold PBS and centrifuged for 5 min at 1500 rpm before being resuspended in ice-cold PBS and kept on ice until analysis. Flow cytometry was performed using a BD Biosciences FACScan II cytometer (Becton Dickinson, San Jose, CA, USA). At least 10,000 cells were collected.
8
EdU Labeling and Fluorescence Microscopy
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Cells were cultured with cell medium containing a final concentration of 10 μM EdU (Keygen, China) for 2 h. They were then fixed in 4% paraformaldehyde for 15 min, and incubated with 0.5% Triton X-100 for 20 min at room temperature. Cells were subsequently washed twice with PBS containing 3% BSA and then reacted with Click-iT for 30 min. The nuclei were stained with Hoechst 33342 (1:2000, Keygen, China) for 15 min. Finally, the images were captured under fluorescence microscopy and the EdU-positive cells were calculated.
9
Hoechst 33342 Apoptosis Detection
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Morphological detection of apoptotic cells was observed using Hoechst 33342 staining. H9C2 cells were plated in six-well plates. After transfection, cells were harvested and washed twice with PBS. Ten microliter of Hoechst 33342 (10 μg/ml; Keygen Biotech, Nanjing, China) was added to each well and incubated for 30 min at 37°C protected from light, and then imaged using an inverted fluorescence microscope. Each treatment was performed in triplicate.
10
Seed Meal Transglutaminase Hydrogel for Cancer
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