DC 2.4 cells were planted on 12-well plates at 1 × 105 cells per well and incubating for 24 h. Bone marrow-derived dendritic cells (BMDCs) were isolated from C57BL/6 mice and cultured in RPMI-1640 medium containing GM-CSF (200 U/ml), with regular replenishment of fresh medium on Day 2 and Day 4 [7 (link),31] (link). On Day 6, BMDCs were collected by centrifugation and re-seeded in 12-well plates. DC 2.4 cells or BMDCs were incubated with NBD-PE-labeled CM and NBD-PE-labeled CM@CaPyro NGs at different times, different concentrations or at different temperatures. Then the cells were collected and analyzed by flow cytometry. For confocal experiments, the cells were washed 3 times with PBS and incubated with 50 mM Lyso Tracker Red DND-99 (Invitrogen, California, America) for 30 min to label lysosome and 1 × Hoechst 33,342 Staining Solution for Live Cells (Beyotime Biotechnology, Shanghai, China) to label nucleus. Then the cells were imaged under confocal microscopy.
Hoechst 33342 staining solution for live cells
Manufactured by Beyotime
Sourced in China
Hoechst 33342 Staining Solution for Live Cells is a fluorescent dye that binds to the minor groove of DNA. It is commonly used to stain the nuclei of live cells for visualization and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dcfh da fluorescent probe, by Beyotime (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Poly ethylene glycol diacrylate, by Merck Group (1 mentions)
Glycidyl methacrylate, by Merck Group (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
Sulfobiotics hsip 1 da, by Dojindo Laboratories (1 mentions)
Lyso tracker red, by Beyotime (1 mentions)
Lsm 980 confocal microscope, by Zeiss (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Nystatin, by Merck Group (1 mentions)
Anti trail, by Cell Signaling Technology (1 mentions)
Nuc cyto mem preparation kit, by Applygen (1 mentions)
Linear pei 25 k, by Polysciences (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Plasmid maxi kit, by Qiagen (1 mentions)
Luciferase assay system kit, by Promega (1 mentions)
Rhodamine b dextran, by Thermo Fisher Scientific (1 mentions)
9 protocols using hoechst 33342 staining solution for live cells
1
Uptake of Nanoparticles by Dendritic Cells
2
ROS assessment in HL-1 cells
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According to the manufacturer’s protocol, a ROS assay kit was used to assess ROS level in HL-1 cells. In short, the cells were incubated with culture medium containing DCFH-DA fluorescent probe and hoechst 33,342 staining solution for live cells (Beyotime, Jiangsu, China) reagent for 30 min at 37 °C in the dark. The samples were examined with a fluorescence microscope. Then the fluorescence intensity was further analyzed using ImageJ software.
3
Polylactidic Acid and Hyaluronic Acid Biocomposite
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Polylactidic acid (PLA) (Mw = 100 kDa, Mw/Mn = 1.85) was purchased from Jinan Daigang Co. (China). Fermentation-derived hyaluronic acid (HA) (sodium salt, Mw = 0.5 MDa) was purchased from Yuancheng Technology Co. (Wuhan, China) and used without further purification. Recombinant Human IL-10 (h-IL-10) and Recombinant Murine IL-10 (m-IL-10) were purchased from PeproTech (USA). Hoechst 33,342 Staining Solution for Live Cells was purchased from Beyotime Biotechnology (Shanghai, China). RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA (1 × ), Penicillin-Streptomycin liquid, phosphate-buffered saline (PBS, pH 7.4) were purchased from Gibco (USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich (USA). All other reagents were obtained from Dai Xuan Biological Technology Co. LTD (China) unless otherwise indicated.
4
Biomimetic Hydrogel Intraocular Lens Fabrication
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2-(2-Ethoxyethoxy) ethyl acrylate (EA), 2,2'-Azobis (isobutyronitrile) (AIBN), poly (ethylene glycol) diacrylate (PEGDA), sodium borohydride (NaBH4), and glycidyl methacrylate (GMA) were purchased from Sigma-Aldrich. N, N-Dimethyl-formamide (DMF), 2-aminoterephthalic acid (NH2-BDC), iron chloride hexahydrate (FeCl3· 6H2O), ascorbic acid,1,10-phenanthroline, and gold chloride trihydrate (HAuCl4 3H2O) were obtained from Aladdin. Commercial hydrophobic acrylate foldable IOLs were bought from Suzhou 66 Vision Tech Co., Ltd (66VT®, FV- 60A, the diameter of the optical region was 6 mm). Cell culture medium DMED/F12 (1: 1), fetal bovine serum (FBS), trypsin, penicillin, and streptomycin were acquired from Giboc. Cell counter kit-8 (CCK-8) and phosphate buffer saline (PBS) were bought from Invitrogen. Hoechst 33342 staining solution for live cells and mitochondrial membrane potential detection kit (JC-1) were obtained from Beyotime Biotechnology. All the eye drops used on animals after the operation were purchased from the Eye Hospital of Wenzhou Medical University.
5
Intracellular H2S Quantification
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Intracellular H2S level was measured using the sensitive fluorescent probe SulfoBiotics-HSip-1 DA (Cat#SB22, DOJINDO, Japan) according to the manufacturer's protocols. Briefly, treated cells were washed and incubated in 5 μM HSip-1 DA working solution for 30 min at 37 °C. Hoechst 33342 staining solution for live cells (Cat#C1027, Beyotime Biotechnology, China) was used for counterstaining the nucleus. All images were obtained with Nikon Eclipse Ti fluorescence microscope (Nikon, Japan) and analyzed using Image J software.
6
Lysosomal Quantification in Cell Lines
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Lyso-Tracker was used to assess the numbers of lysosomes in living cells following the manufacturer’s instructions. KGN and COV434 cells were seeded on confocal dishes for 24 h for attachment and then cultured for another 48 h. Then, the cells were incubated in the dark with 50 nM Lyso-Tracker Red (Beyotime) at 37 °C for 30 min. Finally, the cells were incubated with Hoechst 33342 Staining Solution for Live Cells (Beyotime) in growth medium for 10 min to achieve nuclear DNA staining. The fluorescence intensity was observed under a LSM 980 confocal microscope (Zeiss), and image analysis was performed with ImageJ software.
7
Plasmid Transfection and Cellular Assays
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Linear PEI25K was purchased from Polysciences (USA). Chlorpromazine, nystatin, and cytochalasin D were purchased from Sigma-Aldrich (USA). Nuc-Cyto-Mem Preparation Kit was purchased from Applygen Technologies (Beijing, China). The plasmids of peGFP-N1 encoding enhanced green fluorescent protein, pRFP encoding red fluorescent protein, and pGL4.13 encoding luciferase were propagated in a DH5-strain of E. coli and purified by using a QIAGEN Plasmid Maxi Kit (Germany). YOYO-1, LysoTracker Red DND-99, DIO, dextran rhodamine B (MW 70 kDa) were purchased from Invitrogen (USA). The 5-TAMRA-SE was purchased from MedChem Express (USA). Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Thermo Fisher Scientific (USA). Hoechst 33342 Staining Solution for Live Cells, DAPI staining solution, RIPA lysis buffer, 0.25% trypsin-EDTA, BCA microplate protein assay kit, and ROS detection kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Luciferase Assay System Kit was purchased from Promega (USA). Anti-DR5, anti- TRAIL, and anti-GAPDH were purchased from Cell Signaling Technology (USA). All of the other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Branch PEI25K was purchased from Sigma-Aldrich (USA).
8
Apoptosis Detection in H9c2 Cells
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Mito‐Tracker Red CMXRos (Cat. No. C1049B, Beyotime Biotechnology) and Hoechst 33342 Staining Solution for Live Cells (Cat. No. C1028, Beyotime Biotechnology) were used to identify the apoptotic cells. After different treatments, H9c2 cells were incubated with DMEM containing 200 nM Mito‐Tracker Red CMXRos and 1X Hoechst 33342 Staining Solution at 37℃ for 30 min in the dark. Fluorescence images were scanning using a fluorescence microscope (Leica, Germany).
9
Visualizing Subcellular Localization
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After transfection, cells were incubated for 24 h, then the nucleus was stained with Hoechst 33342 Staining Solution for Live Cells (Beyotime) for 10 min, imaged under a Nikon C2 confocal microscope. GFP fusion proteins were excited with blue light and the nucleus stained with Hoechst 33342 was excited with UV light. The images of cells excited with individual fluorescent channels were taken separately and merged afterward with Nikon imaging software.
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