For immunocytochemical analysis, we fixed the cells with 4% paraformaldehyde for 10 min, and permeabilized it with a PBS solution containing 0.1% of Triton-X 100 for 10 min. After blocking with 5% FBS, we incubated the cells with anti-CD31 (1/30; 14-0319-82, Thermo Fisher Scientific, Waltham, MA, USA) primary antibody at 4°C overnight. Afterward, we incubated the cells with secondary Goat anti-Mouse Alexa Fluor 488 antibody (1/400; A-11001, Thermo Fisher Scientific) for 60 min at room temperature, and we stained the nuclei with Hoechst 33 342 (H342, Dojindo Molecular Technologies, Kumamoto, Japan). We developed the images by IN Cell Analyzer 6000 (GE Healthcare, Chicago, IL, USA). For flow cytometry, we suspended the cells in HBSS containing 3% FBS and 0.3 mM EDTA, fixed with 4% paraformaldehyde, and stained with the same antibodies used in immunofluorescence. Mouse IgG isotype control (401 401, BioLegend, San Diego, CA, USA) was applied as a negative control. We analyzed the cells via a BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA), and performed the data analysis using FlowJo v10 software (BD Biosciences).
Hoechst 33342 h342
Manufactured by Dojindo Laboratories
Sourced in Japan
Hoechst 33342 (H342) is a fluorescent dye commonly used for nuclear staining in biological applications. It selectively binds to the minor groove of double-stranded DNA, emitting a blue fluorescence upon excitation. The dye can be used for DNA quantification, cell counting, and cell cycle analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bz x700 microscope, by Keyence (2 mentions)
Alexa fluor 488 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Mouse igg isotype control, by BioLegend (1 mentions)
Anti cd31, by Thermo Fisher Scientific (1 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
Facscanto 2, by BD (1 mentions)
Fv31s sw, by Olympus (1 mentions)
Fv3000 confocal microscope, by Olympus (1 mentions)
Senescence β galactosidase activity assay kit, by Cell Signaling Technology (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Dfc420, by Leica (1 mentions)
Paraformaldehyde phosphate buffer solution, by Nacalai Tesque (1 mentions)
Pepsin, by Promega (1 mentions)
F1002, by Panagene (1 mentions)
6 protocols using hoechst 33342 h342
1
Immunocytochemistry and Flow Cytometry for CD31
2
Quantifying Lysophagy via Acidic mKeima
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TMEM192-mKeima expressing cells were precultured overnight in the aforementioned DMEM on a glass-bottomed dish (D11130H; Matsunami Glass). To induce lysophagy, cells were treated with 1 mM LLOMe, followed by culture in DMEM (040-30095; Fujifilm)-containing Hoechst 33342 (H342; Dojindo) without LLOMe. After incubation, the cells were subjected to fluorescence microscopy. The cells were examined using a 60× 1.3 NA oil immersion objective and an FV3000 confocal microscope (Olympus) operated using FV31S-SW (version 2.3.1.163) at room temperature. The cells were observed under neutral and acidic conditions using excitation wavelengths of 440 and 594 nm, respectively. The number of puncta under 594 nm excitation was determined using Fiji (National Institutes of Health; https://imagej.net/Fiji ).
First, the maximum signal at 445 nm was identified, and the intensity and position of each signal were measured. The intensity of each signal at 594 nm was then measured at the same position, and the value at 594 nm was compared with the value at 445 nm. If this ratio was above the set criteria, the signal was considered to indicate “acidic mKeima punctum.” Lysophagic activity in the cell in each experiment was quantified according to the above criteria.
First, the maximum signal at 445 nm was identified, and the intensity and position of each signal were measured. The intensity of each signal at 594 nm was then measured at the same position, and the value at 594 nm was compared with the value at 445 nm. If this ratio was above the set criteria, the signal was considered to indicate “acidic mKeima punctum.” Lysophagic activity in the cell in each experiment was quantified according to the above criteria.
3
Quantifying Senescence-Associated β-Galactosidase Activity
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The Senescence β-Galactosidase Activity Assay Kit (fluorescence, plate-based; #23833; Cell Signaling Technology, Danvers, MA, USA) was used for SAβgal staining according to the manufacturer’s protocol. Cells were stained overnight at 37° C in a room CO2 incubator air. The cells were washed with phosphate-buffered saline (PBS) (−) and stained with Hoechst 33342 (H342; Dojindo, Kumamoto, Japan). Imaging and quantification of stains were performed using a BZ-X700 microscope (Keyence, Osaka, Japan).
4
Immunofluorescent Detection of CCR4 in Organs
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The brain, heart and lung were processed for making paraffin section. Slices were immersed in antigen retrieval at over 95 °C for 10 min in a microwave and cooled naturally. After blocking with 5% BSA, the tissues were incubated with primary antibody to CCR4 (ab216560, Abcam, Cambridge, UK; for IF, 1:200 dilution) overnight at 4 °C. Following three washes in PBST (0.1% Tween-20 in PBS), the slices were incubated with a secondary antibody Alexa 488-conjugated donkey anti-rabbit (A21206, Invitrogen, Eugene, OR, USA; 1:500 dilution), and nuclei were stained with Hoechst 33342 (H342, DOJINDO, Kumamoto, Japan; 1:1000 dilution). The number of positive cells was counted in ten randomly selected images under an upright fluorescence microscope (Leica DFC420, Wetzlar, Germany) in a blinded manner, and the results were expressed as the number of positive cells per image (400×).
5
Analyzing Cell Nuclei Morphology with Vibration
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The morphologies of cell nuclei with and without vibrational stimulation were observed from fluorescent images of the cells. After removing the medium, cells were washed three times with 1 mL PBS. Next, cells were fixed with 4% paraformaldehyde (4% – Paraformaldehyde Phosphate Buffer Solution, Nacalai Tesque, Inc., Kyoto, Japan) in PBS for 10 min, washed with PBS, permeabilized with 0.1% Triton X‐100 (Triton X‐100 laboratory grade, Sigma‐Aldrich, MO, USA) in PBS for 5 min, and finally washed with PBS. After removing the PBS, the cells were stained with 0.05% Hoechst 33342 (H342, Dojindo Laboratories, Kumamoto, Japan) for 30 min and again washed with PBS. Then, fluorescence images of the cells were captured using the inverted microscope.
The captured fluorescent images were analyzed by fitting an ellipse to each cell nucleus using Image J. 300 cell nuclei near the gap edge were randomly selected to be analyzed. The angle of each cell nucleus was measured as the angle between the major axis of the fitted ellipse and a straight line parallel to the gap. The aspect ratio of each nucleus was measured as the ratio of the major axis to the minor axis of the fitted ellipse.
The captured fluorescent images were analyzed by fitting an ellipse to each cell nucleus using Image J. 300 cell nuclei near the gap edge were randomly selected to be analyzed. The angle of each cell nucleus was measured as the angle between the major axis of the fitted ellipse and a straight line parallel to the gap. The aspect ratio of each nucleus was measured as the ratio of the major axis to the minor axis of the fitted ellipse.
6
Telomere Length Analysis in Fibroblasts
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Fibroblasts at PD10 were treated with the colcemid kit (Chromocenter, Tottori, Japan) and fixed in Carnoy’s solution following the manufacturer’s protocol. The fixed cells on coverslips were treated with ribonuclease (312-01931, Nippon Gene, Tokyo, Japan) and 0.005 % pepsin (V1959, Promega, WI, U.S.), hybridized with peptide nucleic acid oligonucleotide probes (F1002, Panagene, Daejeon, South Korea), and immuno-stained with Hoechst 33342 (H342, Dojindo, Kumamoto, Japan), according to the manufacturer’s protocols. Images were recorded using a BZ-X700 microscope (Keyence, Osaka, Japan). Quantification was performed using the Telometer (https://demarzolab.pathology.jhmi.edu/telometer/ ), as previously described [46 (link), 47 (link)].
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