The cells were subjected to overnight growth on a 48-well plate in preparation for the EdU incorporation experiment. Following that, the cells were grown in a medium containing the EdU working solution (Beyotime, #C0075). Following a period of incubation at a temperature of 37 °C for a duration of 2 h, the cells were subjected to fixation using a 4% solution of paraformaldehyde (Beyotime, Shanghai, #P0099). Subsequently, permeabilization of the cells was achieved by treating them with a 0.3% solution of Triton X-100 in phosphate-buffered saline (PBS). After the application of the Click Additive Solution, the cells were subjected to incubation with Hoechst dye (Beyotime, #C1025). Subsequently, the enumeration of positive cells was conducted utilizing a fluorescent microscope.
Hoechst dye
Manufactured by Beyotime
Sourced in China
Hoechst dye is a fluorescent stain used in laboratory applications for the detection and visualization of DNA. It is a cell-permeable dye that binds to the minor groove of DNA, emitting a blue fluorescent signal when excited by ultraviolet or blue light. Hoechst dye is commonly used in cell biology, flow cytometry, and various DNA analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Edu working solution, by Beyotime (1 mentions)
Tcs sp5, by Leica (1 mentions)
Dmi3000b, by Leica (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Beyotime (1 mentions)
Click reaction solution, by Beyotime (1 mentions)
P0098, by Beyotime (1 mentions)
P0097, by Beyotime (1 mentions)
C1056, by Beyotime (1 mentions)
Exoquick exosome precipitation solution, by System Biosciences (1 mentions)
10 protocols using hoechst dye
1
EdU-based Cell Proliferation Assay
2
Immunofluorescence Visualization of Cx43 and Golgi
3
Immunofluorescence Analysis of PD-1 and PD-L1
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When the cell density reached 60% confluence on a round glass slide in a 24-well plate, the indicated inhibitors were added for 48 h. The samples were fixed with 4% paraformaldehyde for 15 min at room temperature, treated with cell permeabilization buffer (PBS with 0.2% Triton X-100) for 10 min at room temperature, and then blocked with blocking buffer (PBS with 1% BSA) for 2 h at room temperature. Primary antibodies were incubated with the cells overnight at 4 °C in the blocking solution. After washing in PBS with 0.25% FBS, secondary antibodies including anti-rabbit Alexa Fluor 488 or 594 dye conjugate (Jackson Laboratories, Bar Harbor, ME, USA) were added and incubated for 2 h at room temperature. Finally, the cells were incubated with Hoechst dye (33342; Beyotime, Nanjing, China) in the dark at room temperature for 10 min. To measure PD-1 and PD-L1 protein interactions, the indicated cells were fixed in 4% paraformaldehyde at room temperature for 30 min and then incubated with recombinant human PD-1-Fc protein (PKSH033554; Elabscience, Houston, TX, USA) for 2 h. The secondary antibody was anti-human Fc conjugated with PE (ab99761; Abcam). Fluorescence signals were detected and captured using a fluorescent microscope (DMI3000B; Leica, Wetzlar, Germany).
4
Fluorescent Imaging of Fixed Cells
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Cells were fixed with 4% formaldehyde and then stained with Hoechst dye (Beyotime, Jiangsu, China) for 15–20 min. Images were captured using a phase-contrast fluorescent microscope (Leica Microsystems; Wetzlar, Germany) at ×400 magnification.
5
EdU-based Cell Proliferation Assay
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EdU cell proliferation assays were carried out in 6-well plates. After 48 h transfection, cells were cultured with EdU (Beyotime Biotechnology, Shanghai, China) solution for 2 h and fixed by 4%paraformaldehyde. After removing 4% paraformaldehyde, cells were washed multiple times with washing solution (Beyotime Biotechnology, Shanghai, China). After removal of the washing solution, the cells were immersed with a permeabilized solution (Beyotime Biotechnology, Shanghai, China) and left at room temperature for 15 min. After removing the permeabilization, the cells were washed multiple times with the washing solution. Click reaction solution (Beyotime Biotechnology, Shanghai, China) is prepared according to the product instructions. Hoechst dye (Beyotime Biotechnology Invitrogen) were used to stain nuclei. The EdU-labeled cells were imaged with confocal microscope. The cells were counted using Image J software (1.53t, NIH, Bethesda, MD, USA).
6
Quantifying DNA Damage via Gamma-H2AX
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Pre-treated cells were washed with phosphate-buffered saline and fixed with fixation solution (P0098, Beyotime, China) for 20 min, followed by permeabilization with permeabilization solution (P0097, Beyotime, China) for 15 min. After blocking with 3% bovine serum albumin (BSA) for 30 min, cells were incubated overnight with gammaH2AX antibody (1:400, 9718, CST, USA), followed by incubation with donkey anti-rabbit fluorescent secondary antibody (1:500, 4412, CST, USA) for 1 h the next day. Nuclei were counterstained with Hoechst dye (1:1000, C1011, Beyotime, China) for 15 min, and fluorescence analysis was performed using a fluorescence microscope (BX63, Olympus, Japan).
7
Fluorescence Microscopy of Hoechst-Stained Cells
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Cells were seeded onto coverslips in 12 well plates (3×104 cells/well). After culture for 24 h, the cells were rinsed in PBS and fixed in 4% paraformaldehyde. Then, the cells were rinsed and stained in Hoechst dye (Beyotime). Images were captured with a fluorescence microscope at 400× magnification.
8
Distinguishing Apoptotic and Necrotic Neurons
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Hoechst dye (33258) and propidium iodide (PI) labeling (C1056, Beyotime Biotechnology) were performed to distinguish between apoptotic and necrotic neurons according to the manufacturer's instructions. In each case, at least four microscopic fields were photographed randomly, with three wells were used for each group.
9
Macrophage Cell Death Assay
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THP-1 macrophages were seeded in 24-well plates and treated as described above. Cells were stained with propidium iodide (8 μg/ml) and Hoechst dye (8 μg/ml) (C0003; Beyotime Biotechnology, Shanghai, China) and imaged using a fluorescent microscope (Olympus, Tokyo, Japan). PI-positive cells suggested cell death.
10
Uptake of Labeled Exosomes in NIH-3T3 Cells
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hucMSC-Exos were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, Beyotime Biotechnology, Shanghai, China). The labeled exosomes were isolated through Exoquick exosome precipitation solution (System Biosciences, Palo Alto, USA) to remove excess dye. Mixtures were co-cultured with NIH-3T3 cells in DMEM for 4 h at 37 °C. Then, the cells were washed thrice with PBS to remove excess dye, co-cultured with Hoechst dye (Beyotime Biotechnology, Shanghai, China) at 37 °C for 1 h, and then observed under a fluorescence microscope.
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