Intracellular microinjections were performed using FemtotipsII, InjectManNI2 and FemtoJet systems (Eppendorf, Hauppauge, NY) as previously reported (Brailoiu et al., 2011 (link)). Pipettes were back-filled with an intracellular solution containing, in mM: 110 KCl, 10 NaCl and 20 HEPES (pH 7.2) or the compounds to be tested (G-1 and inositol 1,4,5-trisphophate). The injection time was 0.4 s at 60 hPa with a compensation pressure of 20 hPa in order to maintain the microinjected volume to less than 1% of cell volume, as measured by microinjection of a fluorescent compound (Fura-2 free acid). The intracellular concentration of chemicals was determined based on the concentration in the pipette and the volume of injection.
Injectman ni2
Manufactured by Eppendorf
Sourced in Germany
The InjectMan NI2 is a microinjector device designed for precise and controlled microinjection applications. It provides a stable and reliable platform for performing microinjections in a laboratory setting. The core function of the InjectMan NI2 is to accurately and precisely deliver small volumes of liquids or solutions into cells or other biological samples.
Automatically generated - may contain errors
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Geneart platinum cas9 nuclease, by Thermo Fisher Scientific (3 mentions)
Repli g single cell kit, by Qiagen (2 mentions)
Dm il led microscope, by Leica (2 mentions)
Sodium butyrate, by Merck Group (2 mentions)
α tubulin, by Merck Group (2 mentions)
Gm130, by BD (2 mentions)
Tgn46, by Abcam (2 mentions)
Ergic 53 p58, by Merck Group (2 mentions)
X tremegene 9 transfection reagent, by Roche (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Femtotips 1, by Eppendorf (2 mentions)
Vertical puller, by Narishige (2 mentions)
Nis elements ar 3, by Nikon (1 mentions)
Halocarbon oil 27, by Merck Group (1 mentions)
Halocarbon oil 700, by Merck Group (1 mentions)
Uplans apo 1.3 na, by Olympus (1 mentions)
Femtotips 2 needles, by Eppendorf (1 mentions)
37 protocols using injectman ni2
1
Microinjection of Intracellular Compounds
2
Intracellular Microinjection Technique for Precise Compound Delivery
3
Visualizing Drosophila Embryo Spindle Dynamics
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Imaging was performed on a Visitron Systems Olympus IX81 microscope with a CSO-X1 spinning disk using a UPlanS APO 1.3 NA (Olympus) 60× objective. Embryos 1–2 hr old were manually dechorionated, aligned in heptane glue on 22 × 50 mm coverslips, and covered with a 1:1 mixture of Halocarbon oil 700 and Halocarbon oil 27) (Sigma). Imaging was performed with 400 ms exposure per slice, with five slices per stack and a constant room temperature of 22°C. Embryos were injected using an Eppendorf Inject Man NI 2 and Femtotips II needles (Eppendorf). The anti-DSpd-2 and anti-Dgt6 antibodies were suspended in injection buffer (100 mM HEPES, pH 7.4, and 50 mM KCl), centrifuged at 13,500 × g for 20 min, and injected at a concentration of either 6 mg/ml (anti-Dgt6 or anti-DSpd-2, high concentration) or 1 mg/ml (anti-DSpd-2, low concentration). For cold-treatment assays, single embryos were imaged until metaphase was reached, at which point they were placed in 50 mm ice-cold Petri dishes and covered with 4°C Halocarbon oil. Following 90 min on ice, embryos were reimaged, with 30 s typically expiring between removal from 4°C and the initiation of imaging. In some cases, embryos were removed following 75 min on ice, injected with antibody, and then placed on ice for another 15 min prior to imaging.
4
Microinjected Fluorescent Protein Labeling of Cerebellar Purkinje Cells
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With aid of the microinjection technique yellow fluorescent protein-actin plasmids (pEYFP-actin; 0.195 μg/μl; custom made) were injected into single juvenile (group 2) and mature (group 3) PC cell nuclei of cerebellar slice cultures. Either sterile glass capillaries (diameter: 0.2–0.5 μm, Femtotips; Eppendorf, Germany) or capillaries (Hilgenberg, Nr. 1403512, Germany; borosilicate glass: 1.5 mm/0.2 mm; Puller: Sutter Instruments P97, USA) were filled with 2 μl of plasmid solution. Using an inverted microscope equipped with long-distance phase-contrast optics (Axiovert 35, Zeiss, Germany), PCs were imaged in slice cultures. During microinjection cultures were kept at 37°C on a heating stage. Application settings of the pressure injection devices (InjectMan NI2 and FemtoJet; Eppendorf) were set to inject with 80 hPa for 0.5s The constant pressure was defined as 60 hPa. After microinjection, nutrient medium was replaced carefully and slice cultures then further incubated for at least 24 h to receive a sufficient signal.
5
Single Consortium Sorting and WGA
6
Osteocyte Mechanosensing under Flow Stress
7
Microinjection of DNA Probes in Mammalian Cells
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Mouse embryonic fibroblast26 (link) (MEF, gift from Dr. Akira Kitamura, Hokkaido University) and HEK293 cells (Riken BRC, Ibaraki, Japan) were maintained in a 5% CO2 humidified atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone Lab., Logan, UT, USA), 1 × 105 U l−1 penicillin G and 100 mg l−1 streptomycin sulfate (Wako, Osaka, Japan). The day before the experiment, cells (early passage) were plated on a 35-mm glass-base dish (AGC Techno Glass, Ltd., Shizuoka, Japan) to 60–70% confluence for live-cell imaging or a 6-well plate (Thermo Fisher Scientific, Waltham, MA, USA) for expression assay. The medium was replaced by phenol red-free medium (Opti-MEM, Life Technologies, Gaithersburg, MD, USA) before confocal imaging. Microinjection of the DNA probe was conducted at the confocal microscope stage by combining Femtojet (Eppendorf, Hamburg, Germany) and Injectman NI2 (Eppendorf) with Femtotip (Eppendorf) as an injection needle. Injection pressure was adjusted to deliver the desired amount of DNA.
8
Chemotactic Sensing in Live Cells
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Chemotactic sensing assays were performed as previously described (Mouneimne et al., 2006 (link)). Briefly, cells were plated on glass-bottom dishes (1.5; MatTek) coated with 10 µg/ml fibronectin (Corning) and allowed to adhere for ∼24 h. Before imaging, imaging buffer was added to cells (full medium supplemented with 10 mM Hepes). A micromanipulator (InjectMan NI2; Eppendorf) was fitted with a glass micropipette (Femtotip; Eppendorf), which was loaded with 10 nM IGF with 3-kD fluorescently labeled Dextran clarified by 0.2-µm filters. A chemotactic gradient was generated using a pressure-regulated microinjection system at 25pi with continuous flow (FemtoJet; Eppendorf). Micropipettes were placed ∼25 µm away from nonleading edges of single cells, and IGF was released from the micropipette for 2 min. Experiments were imaged on a Ti-E inverted microscope, with a 60× Plan Apo 1.40 NA objective, and an ORCA-Flash 4.0 V2 CMOS camera, a motorized stage, and an environmental chamber. Imaging was done at 37°C with ambient 5% CO2. Stimulated cells were imaged by DIC microscopy at a 1-s frame rate for 30 min. Wide-field fluorescence, captured at a 1-min frame rate, was used to visualize the chemotactic gradient. Chemotactic sensing was quantified using the sensing index and turning angles of manual traces done in ImageJ (see Sensing index and turning angles).
9
Magnetic Tweezer Manipulation of Cell-Bead Interactions
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We used a magnetic tweezer device as described in Ref. [11] (link). For measurements, 2×105 cells were seeded overnight in a 35 mm diameter tissue culture dish. Thirty minutes before the experiments, cells were incubated with fibronectin-coated paramagnetic beads of 4.5 µm diameter (Invitrogen). A magnetic field was generated using a solenoid with a needle-shaped core (HyMu80 alloy, Carpenter, Reading, PA). The needle tip was placed at a distance of 20–30 µm from a bead bound to the cell using a motorized micromanipulator (Injectman NI-2, Eppendorf). During measurements, bright-field images were taken by a CCD camera (ORCA ER, Hamamatsu) at a rate of 40 frames/s. The bead position was tracked on-line using an intensity-weighted center-of-mass algorithm. Measurements on multiple beads per well were performed at 37 °C for 1 h, using a heated microscope stage on an inverted microscope at 40× magnification (NA 0.6) under bright-field illumination [10] (link).
10
Visualizing Golgi Apparatus Dynamics
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