Inubg2e zilcs

Manufactured by Tokai Hit

Sourced in Japan

The INUBG2E-ZILCS is a laboratory equipment product. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Eclipse ti e, by Nikon (6 mentions) Csu x1 a1, by Yokogawa (4 mentions) Cfi apo tirf 100 1.49 n a oil objective, by Nikon (4 mentions) Optosplit 3 beamsplitter, by Cairn Research (4 mentions) Metamorph 7, by Molecular Devices (4 mentions) Coolsnap hq2 ccd camera, by Teledyne (3 mentions) Evolve 512 camera, by Nikon (3 mentions) Dmi6000b, by Leica (2 mentions) Fugene 6, by Promega (2 mentions) Eclipse ti microscope, by Nikon (2 mentions) Photometrics evolve 512 emccd, by Teledyne (2 mentions) Nis elements software, by Nikon (2 mentions) Evolve 512 emccd camera, by Teledyne (2 mentions) Eclipse ti2 e, by Nikon (2 mentions) Prime bsi scmos camera, by Teledyne (1 mentions) C shg1, by Nikon (1 mentions) Huygens professional version 18, by Scientific Volume Imaging (1 mentions) Coolsnap hq2, by Nikon (1 mentions) Agarose gel, by Thermo Fisher Scientific (1 mentions) Fugene hd, by Promega (1 mentions)

16 protocols using inubg2e zilcs

TIRF Microscopy for Cellular Imaging

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TIRF microscopy was performed on an inverted microscope Nikon Eclipse Ti-E (Nikon) with a perfect focus system. The setup was equipped with a Nikon CFI Apo TIRF 100x1.49 N.A oil objective, Photometrics Evolve 512 EMCCD (Roper Scientific) or CoolSNAP HQ2 CCD camera (Roper Scientific) and controlled with Metamorph 7.7.5 software. The final magnification was 0.063 μm/pixel, which includes the 2.5x magnification introduced by an additional lens (VM lens C-2.5x, Nikon). The temperature was controlled by a stage top incubator INUBG2E-ZILCS (Tokai Hit). The microscope was equipped with TIRF-E motorized TIRF illuminator modified by Roper Scientific France/PICT-IBiSa, Institut Curie. For excitation we used 491 nm 100 mW Stradus (Vortan), 561 nm 100 mW Jive (Cobolt) and 642 nm 110 mW Stradus (Vortran) lasers. We used an ET-GFP 49002 filter set (Chroma) for imaging proteins tagged with GFP and NBD-PC lipid, an ET-mCherry 49008 filter set (Chroma) for imaging X-Rhodamine labeled tubulin, Rh-PE lipid or mCherry-EB3 and an ET-405/488/561/647 for imaging SiR-tubulin labeled MTs or Hilyte 647 porcine brain tubulin. We used sequential acquisition for triple-color imaging experiments.

Rodríguez-García R., Volkov V.A., Chen C.Y., Katrukha E.A., Olieric N., Aher A., Grigoriev I., López M.P., Steinmetz M.O., Kapitein L.C., Koenderink G., Dogterom M, & Akhmanova A. (2020). Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules. Current Biology, 30(6), 972-987.e12.

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Visualizing Chromatin Dynamics in Prepubertal Oocytes

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Prepubertal DOs were stained for 10 min in M2 medium containing 50 ng/ml Hoechst 33342. Then, the selected NSN-oocytes were transferred to and cultured in 199-1 containing 20 ng/ml Hoechst 33342 in an incubator (INUBG2E-ZILCS; Tokai Hit) and observed under a video microscope (DMI6000B; Leica). Phase contrast and fluorescence images of oocytes were taken at 5-min intervals with a charge-coupled device camera (885 EM; Andor), and the chromatin was pseudo-colored red.

Chen F., Lin J., Sun X., Xiao B., Ning S.F., Zhu S., Wang H.L, & Tan J.H. (2017). Mechanisms by which in vitro meiotic arrest and sexual maturity improve developmental potential of mouse oocytes. Scientific Reports, 7, 15763.

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EB3-GFP Dynamics in HeLa Cells

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HeLa cells were
transfected with EB3-GFP using FuGENE 6 (Promega) according to the
manufacturer’s instructions. Experiments were imaged on a Nikon
Eclipse Ti microscope equipped with a perfect focus system (Nikon),
a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa),
an Evolve 512 EMCCD camera (Photometrics) attached to a 2.0×
intermediate lens (Edmund Optics), a Roper Scientific custom-made
set with 487 nm (150 mW) laser, ET-GFP filter (Chroma), a motorized
stage MS-2000-XYZ, a stage top incubator INUBG2E-ZILCS (Tokai Hit),
and lens heating calibrated for incubation at 37 °C with 5% CO₂. Microscope image acquisition was controlled using MetaMorph
7.7, and images were acquired using a Plan Apo VC 40× NA 1.3
oil objective. Imaging conditions were initially confirmed to minimize
GFP bleaching and phototoxicity in untreated cells. For compound-treated
acquisitions, a compound diluted in prewarmed cell medium was applied
to cells and incubated on cells for at least 1 min before commencing
acquisition. Comet count analysis was performed in ImageJ using the
ComDet plugin (Katrukha 2020, ComDet plugin for ImageJ, v0.5.3, Zenodo,
doi: 10.5281/zenodo.4281064). Blinding was not performed as assay
readout is unbiased (Fiji/ImageJ plugins). Data were analyzed using
Prism 9 software (GraphPad).

Rushworth J.L., Thawani A.R., Fajardo-Ruiz E., Meiring J.C., Heise C., White A.J., Akhmanova A., Brandt J.R., Thorn-Seshold O, & Fuchter M.J. (2022). [5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity. JACS Au, 2(11), 2561-2570.

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Photodamage Assay in Live Cells

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Photodamage assays in cells were performed using spinning disk microscopy. COS-7 cells overexpressing GFP-CSPP-L and β-tubulin-mCherry were imaged using confocal spinning disc fluorescence microscopy on an inverted research microscope Nikon Eclipse Ti-E (Nikon), equipped with the perfect focus system (Nikon), Nikon Plan Apo VC 100× N.A. 1.40 oil objective (Nikon) and a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa). The system was also equipped with ASI motorized stage with the piezo plate MS-2000-XYZ (ASI), Photometrics PRIME BSI sCMOS camera (Teledyne Photometrics) and controlled by the MetaMorph 7.10.2.240 software (Molecular Devices). For imaging, we used 487 nm 150 mW Vortran Stradus 488 (Vortran) and 100 mW 561 nm OBIS (Coherent) lasers, the ET-EGFP/mCherry filter (Chroma) for spinning-disc-based confocal imaging. The final resolution using PRIME BSI camera was 0.063 μm/pixel. To keep the live cells at 37°C, a stage-top incubator model INUBG2E-ZILCS (Tokai Hit) was used. The 355 nm laser (Teem Photonics) of the iLAS pulse system was used to induce photodamage by targeting the laser on the spinning disk microscope in a 1-pixel thick line across MTs in the z-plane under the nucleus at 9–11% laser power to induce damage.

van den Berg C.M., Volkov V.A., Schnorrenberg S., Huang Z., Stecker K.E., Grigoriev I., Gilani S., Frikstad K.A., Patzke S., Zimmermann T., Dogterom M, & Akhmanova A. (2023). CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side. The Journal of Cell Biology, 222(4), e202208062.

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Time-Lapse Analysis of Oocyte Chromatin

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Cumulus-free oocytes were stained in M2 medium containing 50 ng/ml Hoechst 33342 for 10 min. The oocytes were then cultured at 37 °C or 39 °C in HCZB medium containing 20 ng/ml Hoechst 33342 in an incubator (INUBG2E-ZILCS; Tokai Hit) and observed under a video microscope (DMI6000B; Leica). Time-lapse analysis was performed over a total of 3 h period. Phase contrast and fluorescence images of oocytes were taken at 15-min intervals with a charge-coupled device camera (885 EM; Andor), and the chromatin was pseudo-colored red.

Lin J., Chen F., Sun M.J., Zhu J., Li Y.W., Pan L.Z., Zhang J, & Tan J.H. (2016). The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model. Scientific Reports, 6, 28347.

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TIRF Microscopy for Live-Cell Imaging

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TIRFM was performed on an inverted research microscope Nikon Eclipse Ti-E (Nikon) with the perfect focus system (PFS) (Nikon), equipped with the Nikon CFI Apo TIRF 100× 1.49 N.A. oil objective (Nikon), Photometrics Evolve 512 EMCCD (Roper Scientific) and controlled with the MetaMorph 7.7 software (Molecular Devices). Images were projected onto the chip of Evolve 512 camera with intermediate lens 2.5X (Nikon C mount adapter 2.5X). To keep in vitro samples at 30°C, we used stage top incubator INUBG2E-ZILCS (Tokai Hit).
For excitation we used 491nm 100mW Stradus (Vortran), 561nm 100mW Jive (Cobolt) and 642 nm 110 mW Stradus (Vortran). We used ET-GFP 49002 filter set (Chroma) for imaging of proteins tagged with GFP, ET-mCherry 49008 filter set (Chroma) for imaging X-Rhodamine labelled tubulin or mCherry-EB3 and ET-405/488/561/647 for imaging SNAP-Alexa647. For simultaneous imaging of green and red fluorescence, we used the triple-band TIRF polychroic ZT405/488/561rpc (Chroma) and the triple-band laser emission filter ZET405/488/561m (Chroma), mounted in the metal cube (Chroma, 91032) together with Optosplit III beamsplitter (Cairn Research Ltd, UK) equipped with a double emission filter cube configured with ET525/50m, ET630/75m and T585LPXR (Chroma). We used sequential acquisition for triple colour imaging experiments.

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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Live Cell Fluorescence Imaging

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Fluorescence imaging of 2D and 3D live cultures was performed on a Nikon spinning disk-based confocal imaging station described in Supplemental Experimental Procedures using a stage top incubator INUBG2E-ZILCS (Tokai Hit) for 37°C/5% CO2 incubation and 37°C lens heating. Simultaneous two-color imaging was performed using the DV2 two-channel simultaneous-imaging system (Photometrics) equipped with the dichroic filter 565dcxr (Chroma). 2D imaging was performed using Nikon Apo TIRF 100x NA 1.49 oil, Plan Apo VC 60x NA 1.4 or a Plan Fluor 40x NA 1.3 objectives. 3D imaging was performed using a Nikon Apo LWD λS 40x NA 1.15 water immersion objective eventually combined with the Nikon Ti intermediate 1.5x magnification lens.

Bouchet B.P., Noordstra I., van Amersfoort M., Katrukha E.A., Ammon Y.C., ter Hoeve N.D., Hodgson L., Dogterom M., Derksen P.W, & Akhmanova A. (2016). Mesenchymal cell invasion requires cooperative regulation of persistent microtubule growth by SLAIN2 and CLASP1. Developmental cell, 39(6), 708-723.

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Highly Sensitive Live-Cell Imaging with TIRF Microscopy

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TIRF microscopy was performed on Nikon Eclipse Ti2-E with the perfect focus with the Nikon CFI Apo TIRF 100× 1.49 NA oil objective, Prime 95B camera (Photometrics), SOLE laser engine (four lasers: 405 nm, 488 nm, 561 nm, and 638 nm; Omicron) and controlled by NIS-Elements software (Nikon). Images were magnified with a 1.5× intermediate lens on Ti2-E before projected onto the camera. The resulting pixel size is 73.3 nm/pixel. Stage top incubator INUBG2E-ZILCS (Tokai Hit) was used to keep cells at 37°C or in vitro samples at 30°C. Imaging medium (DMEM/F12 supplemented with 10% FBS and 5 U/ml penicillin and 50 µg/ml streptomycin) was prewarmed in a water bath at 37°C.
Optosplit III beamsplitter (Cairn Research Ltd.) was used for simultaneous imaging of green and red fluorescence. Stream acquisition was used for simultaneous imaging of green and red fluorescence in vivo. Sequential acquisition was used for three- or four-color imaging experiments.

Huang J., Liang Z., Guan C., Hua S, & Jiang K. (2021). WDR62 regulates spindle dynamics as an adaptor protein between TPX2/Aurora A and katanin. The Journal of Cell Biology, 220(8), e202007167.

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EB3-tdTomato Imaging of Microtubule Dynamics

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HeLa cells were transiently transfected with EB3-tdTomato (gift from Erik Dent; Addgene #50708) using FuGENE 6 (Promega) according to manufacturer’s instructions (see Supplementary Information for all other imaging protocols). Cells were imaged on a Nikon Eclipse Ti microscope equipped with a perfect focus system (Nikon), a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa) and an Evolve 512 EMCCD camera (Photometrics) with a stage top incubator INUBG2E-ZILCS (Tokai Hit) and lens heating calibrated for incubation at 37 °C with 5% CO₂. Cells were incubated in standard cell culture medium with 0.5% DMSO cosolvent, with or without E-AzTax3MP, for 10 min before microscope image acquisition using MetaMorph 7.7 was begun, with EB3-tdTomato imaging performed at 561 nm (0.17 mW, 300 ms every 4 s). Periods of intracellular-ROI-localised 405 nm illuminations (10 µW, 1 scan every 4 s during 24 s periods) were applied during imaging. Acquisition used a Plan Apo VC 60 × NA 1.4 oil objective. Comet count analysis was performed in ImageJ using the ComDet plugin (E. Katrukha, University of Utrecht, https://github.com/ekatrukha/ComDet).

Müller-Deku A., Meiring J.C., Loy K., Kraus Y., Heise C., Bingham R., Jansen K.I., Qu X., Bartolini F., Kapitein L.C., Akhmanova A., Ahlfeld J., Trauner D, & Thorn-Seshold O. (2020). Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton. Nature Communications, 11, 4640.

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Spinning Disk Confocal Microscopy for 3D Imaging

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Spinning disk confocal microscopy was performed on a Nikon Eclipse Ti microscope equipped with a perfect focus system (Nikon), a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa, Japan), an Evolve 512 EMCCD camera (Roper Scientific, Trenton, NJ) attached to a 2.0X intermediate lens (Edmund Optics, Barrington, NJ), a super high pressure mercury lamp (C-SHG1, Nikon), a Roper scientific custom-ordered illuminator (Nikon, MEY10021) including 405 nm (100 mW, Vortran), 491 nm (100 mW, Cobolt), 561 nm (100 mW, Cobolt) and 647 nm (100 mW, Cobolt) excitation lasers, a set of BFP, GFP, RFP and FarRed emission filters (Chroma, Bellows Falls, VT) and a motorized stage MS-2000-XYZ with Piezo Top Plate (ASI). The microscope setup was controlled by MetaMorph 7.7.5 software. Images were acquired using Plan Fluor Apo VC 60x NA 1.4 oil objective. The 3D image reconstruction was carried out using Huygens Professional version 18.04 (Scientific Volume Imaging, the Netherlands). The temperature was controlled by a stage top incubator INUBG2E-ZILCS (Tokai Hit).

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