The U251 or A172 glioma cells were seeded into 96-well plates at 5 × 103 cells/well and incubated for 12, 24, 36, 48, 72, and 96 h. An amount of 10 μL of CCK8 solution (0.5 mg/mL; Sigma, Saint Louis, MO, USA) was added into each well and incubated at 37 °C for 2 h. Finally, the absorbance was measured at 570 nm to assess cell viability. EdU Apollo 567 Cell Tracking Kit (Rib-oBio, Guangzhou, China) was used for cell proliferation. Hochest 33342 (Sigma, Saint Louis, MO, USA) was used for nuclei staining. The percentage of EdU-positive cells in total cells was calculated based on counting 500 random cells in three independent experiments.
Hochest 33342
Manufactured by Merck Group
Sourced in United States
Hochest 33342 is a fluorescent dye used for DNA staining in biological research applications. It binds to the minor groove of double-stranded DNA and emits a blue fluorescent signal when excited with ultraviolet light. The dye is commonly used for nuclear staining, cell cycle analysis, and cell viability assays.
Automatically generated - may contain errors
Lab products found in correlation
β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Synergy 2, by Agilent Technologies (2 mentions)
Mitotracker, by Thermo Fisher Scientific (2 mentions)
Atp lysis solution, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Cck 8 solution, by Merck Group (1 mentions)
Edu apollo 567 cell tracking kit, by RiboBio (1 mentions)
Caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Sgc 7901, by Shanghai Institute of Biochemistry and Cell Biology (1 mentions)
L α phosphatidylcholine, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Facsaria 2 sorp, by BD (1 mentions)
Biotek synergy 2, by Agilent Technologies (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Cck 8, by Beyotime (1 mentions)
Chemidox xrs, by Bio-Rad (1 mentions)
24 protocols using hochest 33342
1
Cell Viability and Proliferation Assay
2
Plasmid Transfection for Apoptosis Induction
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The plasmid pcDNA 3.1(−)/r‐caspase‐3 was constructed by the investigators 19 . AE was purchased from Jiangxi Tiangong Technology (Jiangxi, China), and dimethylsulfoxide (DMSO), l ‐α‐phosphatidylcholine, cholesterol, 3‐2,5‐diphenyl‐tetrazolium bromide (MTT), and Hochest33342 were purchased from Sigma‐Aldrich Co. (St. Louis, MO). Caspase‐3 antibodies were obtained from Cell Signaling Technology (Danvers, MA) and β‐actin antibodies and the corresponding secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RPMI 1640 medium was purchased from Hyclone (Logan, UT) and fetal bovine serum (FBS) was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). The light‐emitting diode (LED) light was purchased from Chongqingjingyu Laser Biological Company (Chongqing, China). Human gastric cancer cell line (SGC‐7901) was purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China.
3
Investigating Ginseng-based Apoptosis Modulation
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RPMI 1640 Medium and Penicillin-Streptomycin Solution were from Hyclone (USA). FBS was from Tianhang Biological Technology (Hangzhou, China). PBS was from CORNING (NY, USA). 0.25% trypsin and CCK-8 were from Beyotime (Nantong, China). SD (Ginseng, Atractylodes, Poria, Licorice) was from Department of Traditional Chinese medicine of the First Affiliated Hospital of Bengbu Medical College (Bengbu, China). 0.22μm syringe filter was from Millpore (Boston, USA). Hochest 33342 was from Sigma (Saint Louis, USA). Verapamil was from Shanghai Pharmaceutical Group Co., Ltd. (Shanghai, China). AV and PI were from BD (Franklin Lakes, USA). Antibodies against Bax and Bcl-2 were from Abcam (Cambridge, England). Substrate Reagent was from Millpore (Boston, USA). β-actin antibody was from SANTA CRUZ (Dallas, USA). Horseradish peroxidase-labeled goat anti-rabbit IgG was from Biosharp (Hefei, China). Flow cytometers BD FACS ARIA II SORP and BD FACSCalibur: BD (Franklin Lakes, USA). Electrophoresis apparatus and gel imaging system: BioTeK Synergy 2 (BioTeK, USA). Power supply and gel imaging system ChemiDox XRS: Bio-Rad (Hercules, USA).
4
Mitochondrial Staining and ATP Measurement
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For mitochondrial staining, oocytes were incubated in Hepes containing 100 nM Mito Tracker (Cat#: M7521, Invitrogen) and 10 µg/mL Hochest 33 342 (Sigma) for 30 min. Images were taken with an Andor Revolution spin disc confocal workstation.
For ATP measurements, the oocytes were first lysed with 100 µl ATP lysis solution (Cat#: S0026, Beyotime) on ice. The samples were then detected by enzyme‐labelled instrument Synergy2 (BioTek) to evaluate ATP level.
For ATP measurements, the oocytes were first lysed with 100 µl ATP lysis solution (Cat#: S0026, Beyotime) on ice. The samples were then detected by enzyme‐labelled instrument Synergy2 (BioTek) to evaluate ATP level.
5
Visualizing Intracellular Localization of Biomimetic Nanoparticles
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To visualize the intracellular localization of BMs, sulfo-cyanine5 NHS ester (Abcam, ab146459, Cambrige, UK) was used to label BMs. Raw 264.7 cells were incubated with labeled BMs at 37 °C for 3 h. After incubation, Raw 264.7 cell membrane was stained with DIO membrane fluorochrome at 37 °C for 20 min (Beyotime Biotechnology, Shanghai, China), and the cell nucleus was stained with Hochest 33342 (Sigma, St. Louis, MO, USA) at 37 °C for 10 min, and then observed the images by laser confocal microscope (Leica SP8 STED 3X, Mannheim, Germany).
6
Detecting Apoptosis: Annexin V and PI Staining
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To detect apoptosis, cells were incubated with DHTS, propranolol or DMSO in different concentrations for 48 h. Then cells were harvested, washed twice with cold 1 × PBS, and re-suspended in 200 μL binding buffer at the density of 1 × 105cells/mL. Then cells were stained with 5 μL Annexin-V (BD Biosciences)for 10 min in dark condition at room temperature and then stained with 5 μL PI for 1 h. At last, cells were analyzed by flow cytometry. The early apoptosis was evaluated based on the percentage of cells with Annexin V+/PI-, while the late apoptosis was Annexin V+/PI+. The results were indicated as mean values from three independent determinations.
To visualize apoptotic bodies, EOMA cells were exposed to different concentrations of DHTS for 24 h, fixed in 4% paraformaldehyde and stained with 1 ml 10 μg/ml Hochest 33342 (Sigma) for 30 min at 37°C in the dark. After thoroughly washed with PBS, the cells were checked for karyopyknosis under the inverted fluorescence microscope.
To visualize apoptotic bodies, EOMA cells were exposed to different concentrations of DHTS for 24 h, fixed in 4% paraformaldehyde and stained with 1 ml 10 μg/ml Hochest 33342 (Sigma) for 30 min at 37°C in the dark. After thoroughly washed with PBS, the cells were checked for karyopyknosis under the inverted fluorescence microscope.
7
Quantifying Autophagy in Bone Marrow Mesenchymal Stem Cells
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BMMSCs were harvested and coated with 4% paraformaldehyde for 15 min on ice. Femurs were fixed, decalcified and cut into 10 μm thick frozen sections. Cells and bone sections were permeabilized with 0.03% Triton-X100 for 15 mins at room temperature and blocked in 5% BSA at 37°C for 30 min. BMMSCs were incubated with specific primary antibody LC3 (Cell Signaling Technology, USA) and bone sections were stained with LC3 and Sca-1 (Abcam, UK) overnight at 4°C. All samples were then incubated with fluorescent secondary antibodies (Cell Signaling Technology, USA) at room temperature for 2 h after rinsing. Hochest 33342 (Sigma-Aldrich) was used to counterstain cell nuclei at room temperature for 3 min. Stained cells and tissues were observed under a confocal microscope (Olympus).
8
Mitochondrial Mass and Membrane Potential Assay
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For imaging mitochondrial mass or morphology independent of membrane potential [21 (link)], live cells were stained by MitoTracker Green (100 nmol·L− 1, Invitrogen) at 37 °C for 30 min or transfected with MitoRFP reporter (Addgene) for 48 h, with nuclei staining by Hochest 33,342 (Sigma). Mitochondrial structure was observed by transmission electron microscopy [22 (link)]. Cells were treated with JC-1 (10 μg/ml, Sigma) at 37 °C for 20 min, while red fluorescence intensity reflecting Δψm was assessed with a fluorescence microscope. Mitochondrial complex I activity was determined by Mitochondrial Complex I Activity Assay Kit (Sigma).
9
Hoechst 33342 Apoptosis Assessment
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Hoechst 33342 staining was used to detect morphological features of cell apoptosis. SKOV-3 cells were treated with cisplatin or ZD55-MnSOD, or the combination of cisplatin and ZD55-MnSOD. After treating for 72 hours, 1 mg/mL (5 μL) Hochest 33342 (Sigma-Aldrich) was added to the cells for 30 minutes and the results were observed under inverted fluorescence microscope. Untreated cells served as a control.
10
Immunofluorescence Staining of 3D-Cultured Neural Stem Cells
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The differentiation status of the NSCs was also assessed by immunofluorescence staining, in accordance with a slightly modified version of procedures in previously published papers (Cui et al., 2016 (link)). For immunofluorescence staining analysis, cells were incubated with the primary antibodies Tuj1 (1:500,05-549, Upstate), Map-2 (1:400; M1406, Sigma), and GFAP (1:200; MAB360, Millipore) overnight at 4°C. The secondary antibodies were anti-mouse IgG FITC antibody (1:200; 31547, Invitrogen) and anti-rabbit IgG FITC antibody (1:1000; 31635, Invitrogen) diluted in blocking buffer. Nuclei are counter-stained with Hochest 33342 (1:500; Sigma). The fluorescent images of 3D-cultured NSCs were visualized on a Leica TCS SP5 scanning laser confocal fluorescence microscope (Leica Microsystems). The number of immunostained cells was counted in each of three random fields per well and the fluorescence images were selected randomly. Quantification of the immunofluorescence signal was performed using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, United States).
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