Sir 647

Manufactured by New England Biolabs

SiR-647 is a fluorescent dye that can be used for live-cell imaging. It emits light in the far-red region of the spectrum, making it suitable for applications where minimal phototoxicity is a concern.

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Lab products found in correlation

Snap cell block, by New England Biolabs (1 mentions) Snap cell tmr star, by New England Biolabs (1 mentions) Eclipse ti inverted microscope, by Nikon (1 mentions) Csu x1 spinning disk confocal head, by Yokogawa (1 mentions) Perfect focus system, by Nikon (1 mentions)

2 protocols using sir 647

Selective Labeling of Newly Synthesized Histones

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For specific labelling of newly synthesized histones^{52 (link),112 (link)}, cells were grown on glass coverslips and pre-existing SNAP-tagged histones were first quenched by incubating cells with 10 μM of the non-fluorescent substrate SNAP-cell Block (New England Biolabs) for 30 min followed by a 30-min wash in fresh medium and a 2-h chase. The new SNAP-tagged histones synthesized during the chase were fluorescently labelled with 2 μM of the red-fluorescent reagent SNAP-cell TMR star or SiR-647 (New England Biolabs) during a 15-min pulse step followed by 30-min wash in fresh medium. Cells were subsequently permeabilized with Triton X-100, fixed and processed for immunostaining. Cells were irradiated with a UVC lamp before the pulse step. Labelling of total histones was performed by a 30-min pulse with 2 μM of the SNAP-cell SiR-647 reagent (New England Biolabs) followed by 30 min wash in fresh medium.

Fortuny A., Chansard A., Caron P., Chevallier O., Leroy O., Renaud O, & Polo S.E. (2021). Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance. Nature Communications, 12, 2428.

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Live Cell Imaging of Protein Dynamics

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Cells were seeded on fibronectin (10 μg/ml)-coated #1.5 glass-bottom dishes and allowed to spread overnight. The following day, cells were labeled with HaloTag ligand conjugated with JF549 (0.4 – 1 μM; Promega) and/or SNAP ligand conjugated with SiR-647 (0.25 – 1 μM; NEB) for 30 minutes, and subsequently washed twice with DMEM. Prior to imaging, the media was replaced with phenol-red free DMEM supplemented with 20 mM HEPES pH7.4. Time lapse image sequences were acquired on a climate-controlled (maintained at 37°C), fully motorized Nikon Ti-Eclipse inverted microscope with Perfect Focus System, equipped with a 60×, 1.49 NA APO TIRF objective (Nikon) with an additional 1.8× tube lens (yielding a final magnification of 108×; Andor Technology), and an Andor Diskovery illuminator coupled to a Yokogawa CSU-X1 confocal spinning disk head with 100 nm pinholes. Image sequences were recorded using a scientific CMOS camera with 6.5-μm pixel size (pco.edge) at a 3 Hz frame rate.

Noh J., Isogai T., Chi J., Bhatt K, & Danuser G. (2022). Granger-causal inference of the lamellipodial actin regulator hierarchy by live cell imaging without perturbation. Cell systems, 13(6), 471-487.e8.

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