U2OS cells were plated on 35-mm glass bottom petri dishes. At 48 hours before microirradiation, cells were cotransfected with the 53BP1-GFP plasmid and either the lamin B1 plasmid or the empty plasmid. The nuclei were stained with Hoescht (1 μg/ml; Hoescht 33342, 4082, Cell Signaling). Live cell imaging was performed using a PLAN APO 60× (numerical aperture: 1.4) objective on a Nikon A1 confocal laser scanning microscope system attached to an inverted ECLIPSE Ti (Nikon Corp., Tokyo, Japan) maintained at 37°C under 5% of CO2 and 20% O2 atmosphere. The microirradiation was performed for selected regions for 2 s with a 405 nm-laser diode set to 2% of the maximum power. Time-lapse images were acquired every 30 s during 75 min. The analysis of protein recruitment kinetics was performed using the NIS-Elements software (Nikon Corp.) by measuring the change in the fluorescence intensities in the irradiated region over time. Intensity values were normalized by the background level in the same region before microirradiation.
Hoescht 33342
Manufactured by Cell Signaling Technology
Hoescht 33342 is a fluorescent stain used for nucleic acid detection. It binds to DNA and emits blue fluorescence when excited by ultraviolet light. The stain is commonly used in various laboratory techniques, such as cell imaging and flow cytometry, to visualize and quantify cellular DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Pampkt172, by Cell Signaling Technology (2 mentions)
Paccs79, by Cell Signaling Technology (2 mentions)
Mk 8722, by Aobious (2 mentions)
Laminin a c, by Cell Signaling Technology (2 mentions)
Sbi 0206965, by Cayman Chemical (2 mentions)
Lysotracker red, by Thermo Fisher Scientific (2 mentions)
β tubulin, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fugene hd, by Promega (2 mentions)
Nis elements software, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Anti tubulin t5168, by Merck Group (1 mentions)
Anti actin a5441, by Merck Group (1 mentions)
2 deoxyglucose 2 dg, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
3 protocols using hoescht 33342
1
Microirradiation-Induced DNA Damage Response
2
Protein Quantification and Localization Protocol
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2-deoxyglucose (2-DG, Sigma, D6134-250MG) was dissolved in 1x DPBS. SBI-0206965 (Cayman Chemical, 18477) and MK-8722 (Aobious, AOB33226) were dissolved in DMSO (Sigma-Aldrich). Hoescht 33342 (Cell Signaling, 4082S) and puromycin (Sigma-Aldrich, P9620) were dissolved in deionized water. For western blotting and immunostaining, antibodies from Cell Signaling Technologies (Denvers, MA USA) were used at 1:1000 dilution unless otherwise noted: P-ACC S79 (3661), P-AMPK T172 (2535), ACC (3662), AMPKα (2532), GAPDH (5174, 1:10,000), Laminin A/C (4777), LKB1 (3047), β-tubulin (2146, 1:10,000). From Sigma-Aldrich, anti-Actin (A5441) was diluted 1:10,000 and anti-Tubulin (T5168) was diluted 1:5000. From Santa Cruz, CaMKK2 (sc-100364) was diluted 1:500–1:1000. From Pierce, horseradish peroxidase-labeled goat anti-rabbit (PI31460) or anti-mouse (PI31430) was diluted 1:1000–1:10,000. Lipofectamine 2000 (11668019) was purchased from Thermo Fisher, and FuGENE HD (E2311) was purchased from Promega. MitoTracker Red (M22425) and LysoTracker Red (L7528) were purchased from Thermo Fisher and diluted in DMSO.
3
Metabolic Pathway Regulation Assay
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2-deoxyglucose (2-DG, Sigma, D6134-250MG) was dissolved in 1x DPBS. SBI-0206965 (Cayman Chemical, 18477), MK-8722 (Aobious, AOB33226), and CCCP (Fisher Scientific, AC228131000) were dissolved in DMSO (Sigma Aldrich). Hoescht 33342 (Cell Signaling, 4082S) was dissolved in deionized water. For western blotting and immunostaining, antibodies from Cell Signaling Technologies (Denvers, MA USA) were used at 1:1000 dilution unless otherwise noted: P-ACC S79 (3661), P-AMPK T172 (2535), ACC (3662), AMPK⍺ (2532), GAPDH (5174, 1:10,000), Laminin A/C (4777), β-tubulin (2146, 1:10,000). From Sigma-Aldrich, anti-Actin (A5441) was diluted 1:10000 and anti-Tubulin (T5168) was diluted 1:5000. Lipofectamine 2000 (11668019) was purchased from ThermoFisher, and FuGENE HD (E2311) was purchased from Promega.
MitoTracker Red (M22425) and LysoTracker Red(L7528) were purchased from ThermoFisher and diluted in DMSO.
MitoTracker Red (M22425) and LysoTracker Red(L7528) were purchased from ThermoFisher and diluted in DMSO.
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