The assay was performed as previously described (57 (link)). 5 × 105 HCC cells were plated at 6-well, and then cells were treated with rLECT2 in serum-free medium for 24 h. After cell harvesting, HCC cells were suspended in Hanks' Balanced Saline Solution (Gibco) containing with 5% fetal bovine serum (HyClone) and 5 μg/ml Hoechest 33342 (Sigma-Aldrich) for 90 min at 37 °C. In some cases, cells were incubated with Hoechst 33342 in the presence of 50 μM verapamil (Sigma-Aldrich) for reliable gating of SPCs. Subsequently, cells were centrifuged and resuspended in cold PBS containing 1 μg/ml propidium iodide (Sigma-Aldrich). SPCs were analyzed by flow cytometer (Beckman Coulter, Inc). The Hoechst 33342 was excited with the UV laser at 351 to 364 nm, and fluorescence was measured using a 515-nm SP filter (Hoechst blue) and a 608 EFLP optical filter (Hoechst red). A 540 DSP filter was used to separate the emission wavelengths.
Hoechest 33342
Manufactured by Merck Group
Sourced in United States
Hoechest 33342 is a fluorescent dye used in laboratory applications to stain and visualize DNA. 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
Verapamil, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Hank s balanced saline solution, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Oxiselect dna double strand break staining kit, by Cell Biolabs (1 mentions)
Laser scanning confocal microscopy, by Keyence (1 mentions)
Glass bottomed cell culture dishes, by NEST Biotechnology (1 mentions)
Cm3050, by Leica camera (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Facsaria 2, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Anti asc, by Novus Biologicals (1 mentions)
Pyronin y, by Merck Group (1 mentions)
Anti ki67, by BioLegend (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Gelatin, by Adamas (1 mentions)
Chitosan, by Maclin Biochemical Technology (1 mentions)
Dihydroethidium dhe, by Merck Group (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
15 protocols using hoechest 33342
1
Identification of Side Population Cells
2
Assessing DNA Double-Strand Breaks Using Immunofluorescence
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DNA-DSBs formation was examined using the OxiSelect DNA Double-Strand Break Staining Kit (Cell Biolabs, San Diego, United States). The cells (5×105 cells/well) were cultivated on glass-bottomed cell culture dishes (Nest Biotechnology, China) at 37 °C for 24 h. Next, cell cultures were subsequently preincubated with either 50 μM BSO or 5 mM OTC for 20 h prior to exposure to DMAE-CB. Then, the cells were treated with 0.01 mM DMAE-CB in the presence or absence of BSO or OTC at 37 °C for 24 h. For immunofluorescent staining, the cells were fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature, washed with cold PBS (4ºC) once, and incubated with ice-cold 90% methanol for 10 minutes at 4ºC. Then the cells were incubated with blocking buffer for 30 minutes at room temperature on an orbital shaker and washed with cold PBS once. Thereafter, cells were incubated with anti-phospho-Histone (γ-H2AX) Antibody Solution, and incubated for 1 hour. After washed 5 times with Wash Buffer (PBST), the cells were incubated with secondary antibody, Cy3 Conjugate Solution for 1 hour at room temperature. Cells were further incubated with 10 mg/ml Hoechest 33342 (Sigma) for 5 min. Specimens were analyzed by laser scanning confocal microscopy (Keyence Co., Osaka, Japan).
3
Immunohistochemical Analysis of Nerve Regeneration
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The rostral and caudal segments of the grafted guidance channel, along with attached host cord segments were sectioned longitudinally on a cryostat (Leica CM3050, Nussloch, Germany) at 20–25 µm. The sections were rinsed in PBS, blocked, and permeabilized in PBS containing 10% normal goat serum and 0.3% Triton X-100 for 1 hour at room temperature, and incubated in primary antibodies overnight at 4˚C. Monoclonal antibody PhosphoDetect anti-neurofilament H mouse mAb (SMI-31) (1:1000; Chemicon, Temecula, CA, USA), mouse anti rat anti-CD68 (ED-1) (1:100; Millipore Antibodies, Billerica, MA, USA), mouse anti rat glial fibrillary acidic protein (GFAP) (1:300; Sigma-Aldrich), Nerve growth factor receptor (NGFR) P75 (1:200; Sigma-Aldrich) were used to identify axons, macrophages, astrocytes and SCs (Liu et al., 2004, 2007; Titsworth et al., 2007, 2008). On the following day, sections were incubated with either fluorescein-conjugated goat anti-rabbit (1:100, ICN Biochemicals, Aurora, OH, USA) or rhodamine-conjugated goat anti-mouse antibodies (1:100, ICN Biochemicals), and Hoechest 33342 (10 μg/mL, Sigma-Aldrich). Slides were examined with an Olympus BX60 microscope (Olympus America, Inc., Melville, NY, USA). Primary antibody omission and mouse and rabbit isotype controls (Zymed Lab Inc., San Francisco, CA, USA) were used to confirm the specificity of the antibodies.
4
Hoechst 33342 DNA Staining Protocol
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The cells were removed, pelleted by centrifugation, washed with PBS (phosphate-buffered saline), and suspended at 1 × 106 cells/ml in PBS supplemented with 4% FBS and incubated at 37 °C for 60 min in the dark with the DNA binding dye, Hoechest 33342 (5 μg/ml, Sigma, USA), either alone or in the presence of verapamil (500 μM, Sigma, USA). After incubation, 1 μg/ml propidium iodide was added and then the cell suspension was filtered through a 40 μm cell strainer to obtain single suspension cells, and the cells were kept at 4 °C in the dark before FACS. Analyses and sorting were performed using BD FACS Aria II (BD, New Jersey, USA).
5
Hoechst 33342 Staining and FACS Sorting
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4T1-HA cells were suspended at 1 × 106 cells per ml in culture medium and stained with 9.0 μg ml−1 Hoechest 33342 (Sigma-Aldrich, St. Louis, MO) for 90 min at 37 °C (ref. 60 (link)). After washing, cells were analysed and sorted by a triple laser MoFlo (Cytomation, Fort Collins, CO) with Summit software (Cytomation) at Keio GCOE FCM Core Facility (Keio University School of Medicine, Tokyo, Japan). Hoechst 33342 was excited at 350 nm, and fluorescence emission was detected by using a 405/BP30 and 570/BP20 optical filter for Hoechst blue and Hoechst red, respectively, and a 550 nm long-pass dichroic mirror (all Omega Optical Inc.) to separate the emission wavelengths. Both Hoechst blue and red fluorescence are shown on a linear scale. Propidium iodide (PI) fluorescence was measured through 630BP30 after excitation at 488 nm with an argon laser, and a live cell gate was defined that excluded the cells positive for PI. Addition of 15 μg ml−1 reserpine resulted in the complete disappearance of the side population (SP) fraction (Sigma-Aldrich). Isolated SP and main population (MP) were re-suspended in culture medium and cell number and viability were confirmed. Then, cells were diluted to appropriate injection doses, mixed with BD Matrigel (BD Bioscience) according to manufacturer61 (link).
6
Laser Microdissection of GCs in DG
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Frozen tissue was sectioned into 12-μm slices and placed on an MMI Membrane Slide (Molecular Machines & Industries, 50103). Tissue sections were incubated with Hoechest 33342 (Sigma Aldrich, B2261) on ice for 10 min, washed in RNase-Free PBS, and dried. Afterward, the MMI Membrane Slide is inverted and placed onto a glass slide for protection against contamination. GCs in the DG were identified and laser microdissected using MMI CellCut. The isolated cells were collected by MMI Isolation Caps (Molecular Machines & Industries, 50202) for transcriptional analysis.
7
Subcellular Localization of PoDot1-GFP Protein
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Hyphae of PoDot1-GFP strain were observed under a high sensitivity laser scanning confocal microscope (ZEISS LSM780) (Carl Zeiss, Germany). The nuclei were stained in the dark for 15 min by Hoechest 33342 (Sigma, United States). The green fluorescence of PoDot1-GFP was observed by excitation light at 488 nm, and the blue nuclei stained with Hoechest 33342 were observed with excitation light at 405 nm. The subcellular localization of PoDot1 was determined by comparing whether green fluorescence and blue fluorescence overlap.
8
Immunofluorescence Microscopy Protocol
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Cells were fixed on microscope slides with 4% paraformaldehyde in 1× PBS for 30 min., and were permeabilized with 0.1% Triton X-100 in 1X PBS for 5 min. After washing with 1× PBS twice, cells were blocked with 1 mg/ml BSA for 1 h. Subsequent sequential steps included incubation with the primary antibody for 3 h, three washes with 1× PBS, incubation with the secondary antibody for 2 h, three washes with 1× PBS and staining with 1 μg/ml Hoechest 33342 (Sigma) in 1× PBS for 5 min. The slides were mounted, and examined by fluorescence microscopy.
9
Immunofluorescent Imaging of ASC in THP-1 Cells
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THP-1 cells were fixed with 4% paraformaldehyde (PH 7.4) at room temperature for at least 20 min and ruptured with 0.1% Triton-X-100 for 30 min. Non-specific antigens were blocked with a 5% BSA solution. Cells were incubated with anti-ASC (1:50; Novus) at 4 °C overnight and then incubated with diluted fluorescent secondary antibody at 4 °C for 1 h. The nuclei were counterstained with hoechest33342 (1:300; Sigma Aldrich) for 15 min at room temperature, followed by an anti-quenching agent. Images were taken with a confocal laser microscope (Nikon, Tokyo, Japan). Exposure to light was avoided throughout the whole process.
10
Cell Cycle Analysis of CD271-Knockdown Cells
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CD271-knockdown cells were fixed with 70% ethanol at −20 °C. The fixed cells were stained with anti-Ki67 (1:60)(Biolegend) and 10 μg/ml propidium iodide. To perform cell cycle analysis for live cells, the cells were incubated with 5 μg/ml Hoechest 33342 and 0.2 μg/ml pyroninY (Sigma) for 1 h at 37 °C. The stained cells were analyzed by flow cytometry.
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