lu n4 laser unit, by Nikon - Product details

1

Isolation and Transduction of Neonatal Cardiomyocytes

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Neonatal cardiomyocytes (P1, CD1 background; breeding pairs were supplied by Charles River Laboratories, Sulzfeld, Germany) were isolated (Figure 7b) and enriched using the neonatal heart dissociation and isolation kits (Miltenyi) (for details, see: Raulf, Voeltz et al. [35 (link)] and manufacturer’s instructions).
Then, NNCMS were seeded on fibronectin- (0.5 µg/mL, Sigma-Aldrich, Merck, Darmstadt, Germany) coated 96 well glass-bottom plates (Greiner) and cultured in a 20% IMDM medium (30,000 NNCMs/well). After 24 h, FCS was reduced to a 2% IMDM medium (IMDM, 2% v/v FCS, 100 U/mL/100 µg/mL P/S, 0.1 mM NEAA, and 0.1 mM β-ME), and the virus was added to the medium (0.55 Virus Genomes (VG)/cell). Two days post-transduction, the medium was changed (2% IMDM), and three days post-transduction, mCherry⁺ cells were observed. Transduction efficiency was calculated as a ratio of double positive NNCMs (α-actinin⁺ and mCherry⁺)/all NNCMs (α-actinin⁺) in four fields of view/well (Nikon Eclipse Ti and Nikon A1R MP system, Nikon, Tokyo, Japan) with DAPI, Cy3, and Cy5 filters; 20× objective; and Lasers (LU-N4 laser unit, 405 nm, 488 nm, 561 nm, 640 nm) using NIS-Elements AR 5.11.01 software (Nikon).

Niemann P., Schiffer M., Malan D., Grünberg S., Roell W., Geisen C, & Fleischmann B.K. (2022). Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells. Cells, 11(4), 694.

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2

Intracellular Ligand Trafficking Visualization

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Indicated cells were seeded in 18 well glass bottom μ-Slides (Ibidi, Cat. No. 81817) at a density of 15k/well. Fluorescencly labeled ligands were incubated with the cultured cells for 0.25 , 3, 6 or 24 hours, 30 min before image acquisition, cells were additionally incubated with Lysotracker (ThermoFisherScientific, Cat. No. L7528/L7526/L12492) was added for 30 minutes. Fluorescently labeled anti-IGF-2R (Novus Biological, NB300–514AF647) was added for 30 minutes. Cells were washed 3× in PBS and immediately proceeded to imaging.
Confocal laser scanning microscopy was performed on a Nikon A1R HD25 system equipped with a LU-N4 laser unit (Lasers used: 488 nm, 561 nm, 640 nm). Data was acquired using an 20×, NA 0.75, WD 1.00 mm air objective (Plan Apochromat Lambda) in combination with 1 multialkaline (EM 650 LP) and 2 GaAsP detectors (DM 560 LP EM 524/42 (503–545) and DM 652 EM 600/45 (578–623)). Acquisition was controlled via NIS Elements software and data was analyzed via Fiji and custom-written Python Scripts.

Huang B., Abedi M., Ahn G., Coventry B., Sappington I., Wang R., Schlichthaerle T., Zhang J.Z., Wang Y., Goreshnik I., Chiu C.W., Chazin-Gray A., Chan S., Gerben S., Murray A., Wang S., O’Neill J., Yeh R., Misquith A., Wolf A., Tomasovic L.M., Piraner D.I., Gonzalez M.J., Bennett N.R., Venkatesh P., Satoe D., Ahlrichs M., Dobbins C., Yang W., Wang X., Vafeados D., Mout R., Shivaei S., Cao L., Carter L., Stewart L., Spangler J.B., Bernardes G.J., Roybal K.T., Greisen P J.r., Li X., Bertozzi C, & Baker D. (2023). Designed Endocytosis-Triggering Proteins mediate Targeted Degradation. bioRxiv.

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3

Subtilisin-Treated Microtubule Binding Assay

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Paclitaxel-stabilized MTs were prepared containing 10% rhodamine tubulin, 10% biotinylated tubulin, and 80% unlabelled tubulin (Cytoskeleton, Inc.), then treated ± 0.1 mg/ml subtilisin for 30 min to remove predominantly β-tubulin C-terminal tails (CTTs), checked by western blot. The reaction was stopped by the addition of 10 mM pefabloc, and MTs were isolated by centrifugation. GFP-CKK domain binding was analyzed by TIRFM, using flow chambers assembled from plasma-cleaned glass coverslips and microscope slides. Chambers were incubated sequentially with 1 mg/ml PLL-PEG biotin (Susos AG), block solution (1% plurionic F-127, 4 mg/ml casein), 0.5 mg/ml neutravidin, and MTs ± CTTs (as indicated). Each incubation was followed by two washes with MRB80 buffer (80 mM PIPES, 4 mM MgCl2, and 1 mM EGTA [pH 6.8]) supplemented with 80 mM KCl, 20 μM paclitaxel, 4 mM DTT and 2 mg/ml casein. The final binding reaction contained 200 nM CKK-GFP in MRB80 with 80 mM KCl, 20 μM taxol, 4 mM DTT and 2 mg/ml casein and an oxygen scavenger mix (400 μg/ml glucose oxidase, 200 μg/ml catalase). TIRFM was performed on an Eclipse Ti-E inverted microscope with a Perfect Focus System, CFI Apo TIRF 1.49 N.A. oil objective, H-TIRF module and LU-N4 laser unit (Nikon). Images were recorded with a 100 ms exposure on an iXon DU888 Ultra EMCCD camera (Andor) controlled with NIS-Elements AR Software (Nikon).

Atherton J., Jiang K., Stangier M.M., Luo Y., Hua S., Houben K., van Hooff J.J., Joseph A.P., Scarabelli G., Grant B.J., Roberts A.J., Topf M., Steinmetz M.O., Baldus M., Moores C.A, & Akhmanova A. (2017). A structural model for microtubule minus-end recognition and protection by CAMSAP proteins. Nature structural & molecular biology, 24(11), 931-943.

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4

Confocal Laser Scanning Fluorescence Imaging of Liquid Crystal Elastomers

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A confocal laser scanning fluorescence microscope (A1⁺ system, Nikon) was used to obtain confocal PFOM images of polydomain sample at 25 °C (nematic) with the higher 3D optical resolutions. An optically pumped semiconductor laser (LU-N4 Laser Unit, Nikon, equipped with Sapphire 488, Coherent Inc.) was used to excite the fluorescent probes at 488 nm and the emitted light between 525 and 595 nm was collected. An objective lens with an NA of 0.90 (TU PlanFluor100 ×, Nikon) was used. The confocal PFOM images were acquired at the voxel size of typically 30(x) × 30(y) × 50(z) nm³. The excitation laser was linearly polarised and emitted fluorescence in the same polarisation was collected using a polariser. The obtained images were properly deconvoluted using the associated software. In the present LCEs with dyes, image slices from the top surface to a few µm depth were properly taken with sufficient fluorescent intensity.

Ohzono T., Katoh K., Minamikawa H., Saed M.O, & Terentjev E.M. (2021). Internal constraints and arrested relaxation in main-chain nematic elastomers. Nature Communications, 12, 787.

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5

Single-molecule live-cell imaging

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All imaging was conducted on an Eclipse Ti-E inverted microscope with a CFI Apo TIRF 1.49 NA oil objective, Perfect Focus System, H-TIRF module, LU-N4 laser unit (Nikon), and a quad-band filter set (Chroma). Images were captured on an iXon DU888 Ultra EMCCD camera (Andor), controlled with NIS-Elements AR Software (Nikon). The microscope was kept in a temperature-controlled environmental chamber (Okolab). Files were imported into Fiji (ImageJ, NIH) (Schindelin et al, 2012 (link)) and analyzed. Kymographs were generated using KymographClear (Mangeol et al, 2016 (link)).

Mukhopadhyay A.G., Toropova K., Daly L., Wells J.N., Vuolo L., Mladenov M., Seda M., Jenkins D., Stephens D.J, & Roberts A.J. (2024). Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport. The EMBO Journal, 43(7), 1257-1272.

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6

Subtilisin-Treated Microtubule Binding Assay

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Paclitaxel-stabilized MTs were prepared containing 10% rhodamine tubulin, 10% biotinylated tubulin, and 80% unlabelled tubulin (Cytoskeleton, Inc.), then treated ± 0.1 mg/ml subtilisin for 30 min to remove predominantly β-tubulin C-terminal tails (CTTs), checked by western blot. The reaction was stopped by the addition of 10 mM pefabloc, and MTs were isolated by centrifugation. GFP-CKK domain binding was analyzed by TIRFM, using flow chambers assembled from plasma-cleaned glass coverslips and microscope slides. Chambers were incubated sequentially with 1 mg/ml PLL-PEG biotin (Susos AG), block solution (1% plurionic F-127, 4 mg/ml casein), 0.5 mg/ml neutravidin, and MTs ± CTTs (as indicated). Each incubation was followed by two washes with MRB80 buffer (80 mM PIPES, 4 mM MgCl2, and 1 mM EGTA [pH 6.8]) supplemented with 80 mM KCl, 20 μM paclitaxel, 4 mM DTT and 2 mg/ml casein. The final binding reaction contained 200 nM CKK-GFP in MRB80 with 80 mM KCl, 20 μM taxol, 4 mM DTT and 2 mg/ml casein and an oxygen scavenger mix (400 μg/ml glucose oxidase, 200 μg/ml catalase). TIRFM was performed on an Eclipse Ti-E inverted microscope with a Perfect Focus System, CFI Apo TIRF 1.49 N.A. oil objective, H-TIRF module and LU-N4 laser unit (Nikon). Images were recorded with a 100 ms exposure on an iXon DU888 Ultra EMCCD camera (Andor) controlled with NIS-Elements AR Software (Nikon).

Atherton J., Jiang K., Stangier M.M., Luo Y., Hua S., Houben K., van Hooff J.J., Joseph A.P., Scarabelli G., Grant B.J., Roberts A.J., Topf M., Steinmetz M.O., Baldus M., Moores C.A, & Akhmanova A. (2017). A structural model for microtubule minus-end recognition and protection by CAMSAP proteins. Nature structural & molecular biology, 24(11), 931-943.

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Lu n4 laser unit

Lab products found in correlation

6 protocols using lu n4 laser unit

Isolation and Transduction of Neonatal Cardiomyocytes

Intracellular Ligand Trafficking Visualization

Subtilisin-Treated Microtubule Binding Assay

Confocal Laser Scanning Fluorescence Imaging of Liquid Crystal Elastomers

Single-molecule live-cell imaging

Subtilisin-Treated Microtubule Binding Assay

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