To detect the effect of nanofibers on scavenging intracellular ROS, DCFH-DA staining (Beyotime, China) was used. Briefly, after RAW 264.7 cells cocultured with LPS and nanofibers for 24 h, DCFH-DA was used to incubate the samples for 30 min, then the live RAW 264.7 cells were stained by using Hoechst 33342 solution (Beyotime, China). For the blank group, RAW 264.7 cells were added to the plates. The results were detected by laser scanning confocal microscope at 485 nm.
Hoechst 33342 solution
Manufactured by Beyotime
Sourced in China
Hoechst 33342 solution is a fluorescent dye commonly used in laboratory research. It is a blue fluorescent dye that binds to the minor groove of DNA. The solution can be used to stain and visualize DNA in various applications such as flow cytometry, fluorescence microscopy, and cell-based assays.
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Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Tunel assay kit, by Beyotime (3 mentions)
Paraformaldehyde (pfa), by Beyotime (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Edu solution, by Thermo Fisher Scientific (2 mentions)
Dcfh da staining, by Beyotime (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Dcfh da assay, by Beyotime (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Mitochondrial membrane potential detection kit, by Keygen Biotech (1 mentions)
Annexin 5 fitc pi, by Keygen Biotech (1 mentions)
Glucose solution, by Thermo Fisher Scientific (1 mentions)
Trypan blue powder, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
23 protocols using hoechst 33342 solution
1
Nanofiber-Mediated ROS Scavenging in Macrophages
2
ROS Detection in PDLSCs
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DCFH-DA assay (Beyotime, Shanghai, China) was used to detect the levels of ROS in PDLSCs. After plating the cells onto 24-well culture dishes (2 × 104/well) overnight, the cells were treated with tFNAs or LPS for 6 h, followed by incubation with a DCFH-DA probe for 20 min. To locate the cell nucleus, Hoechst 33342 solution (Beyotime, Shanghai, China) was used to stain the live cells. Finally, the sample images were captured using an inverted fluorescence microscope (DMi8, Leica, Wetzlar, Germany).
3
Warangalone Induces Autophagy-Mediated Apoptosis
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Warangalone was purchased from Shanghai Yuanye Bio-Technology (Shanghai, China); molecular formula, C25H24O5; molecular weight, 404.462; analysis of standard substance by high-performance liquid chromatography (HPLC) ≥ 98%. Dulbecco's modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin, trypsin, and TRIzol were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 4',6-diamidino-2-phenylindole (DAPI), monodansylcadaverine (MDC), dimethyl sulfoxide (DMSO), 3-methyladenine (3-MA) and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against GAPDH, LC3, BAX, BCL-2 and PARP were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against VDAC1 and PINK1 were purchased from ABclonal Technology (Boston, MA, USA). Antibodies against P62 and Parkin were purchased from Proteintech (Chicago, IL, USA). Hoechst 33342 solution was obtained from Beyotime Biotechnology (Shanghai, China). Cell cycle detection, reactive oxygen species (ROS) analysis, Annexin V-FITC/PI, and mitochondrial membrane potential detection kits were purchased from KeyGEN BioTECH (Nanjing, Jiangsu, China). MitoTracker Red CMXRos and LysoTracker™ Green DND-26 were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
4
Amyloid-β Peptide Inhibition Assay
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Synthetic human Aβ (1-42) peptides (Aβ42) were purchased from Chinapeptide (Shanghai, China). The DAPK1-specific inhibitor (4Z)-4-(3-pyridylmethylene)-2-styryl-oxazol-5-one (C6) was acquired from Calbiochem (California, United States). The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (tanespimycin, 17-AAG) was from Macklin (Shanghai, China). DMSO, trypan blue powder, 1, 1, 1, 3, 3, 3-hexafluoro-isopropanol (HFIP), cycloheximide (CHX), poly-D-lysine and cytosine β-D-arabinofuranoside were purchased from Sigma-Aldrich (Missouri, United States). 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nonfat milk powder and bovine serum albumin (BSA) were from Sangon (Shanghai, China). Hoechst 33342 solution was purchased from Beyotime (Shanghai, China). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), sodium pyruvate, GlutaMAX™ Supplement, Hank's balanced salt solution (HBSS), glucose solution, trypsin and penicillin-streptomycin were provided by Gibco (Texas, United States). Neurobasal medium and the B-27 supplement were obtained from BasalMedia (Shanghai, China). The anti-phospho-Ser71-Pin1 (pS71-Pin1) antibody was prepared and purified by immunizing rabbits with a peptide sequence (CSQSRRPSSWR) of Pin1 containing the pS71 residue (Abmart, Shanghai, China).
5
Sirtinol-Induced Apoptosis Mechanisms
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SAL, Sirtinol and 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Hoechst 33342 solution, the BCA protein assay kit, radioimmunoprecipitation (RIPA) lysis buffer and BeyoECL Plus were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The caspase-3 enzyme-linked immunosorbent assay (ELISA) kit was purchased from the Nanjing Jiancheng Bioengineering Research Institute (cat no. H076; Nanjing, China). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit was obtained from Sigma-Aldrich; Merck KGaA. Antibodies against SIRT1 (cat no. 9475), phosphorylated (p)-FOXO3α (cat no. 9466), B-cell lymphoma 2 (Bcl-2; cat no. 3498) and Bcl-2 associated X protein (Bax; cat no. 2772) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH (cat no. 10494-1-AP) and secondary antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).
6
Immunofluorescent Staining Protocol for Cells
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Cells were seeded in 12-well plates in which glass coverslips were placed, and immunofluorescent staining was performed when cells grew to approximately 90% confluency. After 20 min fixation using 4% paraformaldehyde, the cell membrane was permeabilized with PBS solution containing 0.5% Triton X-100 (Beyotime Biotechnology, China) for 20 min, and after 2 hr blocking with 5% BSA, cells were incubated with one of the indicated primary antibodies at 4°C overnight in a humidified chamber. Then, following a triple wash, the cells were exposed to the Alexa Fluor 488 conjugated secondary antibody for 1 hr in a dark environment. The cell nuclei were then counterstained using Hoechst 33342 solution (Beyotime Biotechnology, China) for a duration of 10 min. Finally, images were visualized and captured using fluorescence microscopy.
7
Apoptosis Induction in BUMPT Cells
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BUMPT cells (generated by Drs. Wilfred Lieberthal and John Schwartz and obtained from Dr. Zheng Dong, Augusta University, Augusta, GA) were treated with cisplatin or/with sTim-3 protein (80 μg/mL) (with or without NF-κB inhibitors TPCA1 (50 μM) or PDTC (40 μM)) for 24 h. Apoptotic cells were analyzed by staining with Hoechst 33342 solution (10 μg/mL; Beyotime Biotechnology, China), and then observed with Nikon inverted microscope (NIKON A1R Storm) and calculated by ImageJ software.
8
Click-iT EdU Cell Proliferation Assay
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The cell proliferation assay was performed using Click‐iT™ EdU Cell Proliferation Imaging Kit (C10340, Thermo Fisher). The cells were plated in 96‐well plates at 7 × 103 cells, treated for 2 h at 37°C with 50 mol/L EdU solution, fixed for 30 min using 4% paraformaldehyde, and treated with Triton X‐100 (Thermo Fisher) for 10 min. The cells were reacted for 30 min with Click‐iT reaction solution in the dark before nuclear staining using Hoechst33342 solution (Beyotime). The cell proliferation rate was assessed using the images taken under a fluorescent microscope (Keyence).
9
Quantifying Apoptosis in Ovarian Cancer Cells
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The apoptosis of SKOV-3 and OV-90 cells with different transfections was confirmed by Hoechst 33342 staining according to previous report [25 (link)]. SKOV-3 and OV-90 cells were seeded in culture dish, added with 10 μL Hoechst 33342 solution (Beyotime Biotechnology, Nantong, China), and cultured for 10 min at 25 °C. A fluorescence microscopy (Olympus, Tokyo, Japan) was used to observe the changes in morphology of SKOV-3 and OV-90 cells (chromatin condensation, fragmentation and cell shrinkage). The apoptotic cancer cells were counted from 400 cells in 12 fields/well, and apoptosis rate (%) = apoptotic cancer cells/total cancer cells× 100.
10
Apoptosis Assay in RAOECs
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RAOECs (1×105 cells/well in 12-well plates) were treated with TNF-α or transfected with different constructs. After washing with PBS, the cells were incubated with 2.5 µl Hoechst 33342 solution and PI (Beyotime Institute of Biotechnology) for 0.5 h in the dark at 4°C. After washing 3 times with PBS, the stained cells were imaged using a DMi8 inverted fluorescence microscope at 200× magnification (Leica Microsystems GmbH).
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