The preparation of the AVIFJ nozzle was described in previously published articles [36] (link). Briefly, the nozzle was fabricated by pulling glass micropipettes (BF 150-110-10; Sutter, USA) with a puller (P-1000; Sutter, USA). A micro-forge needle instrument (MF-900, Narishige, Japan) was used to cut the needle to a designated caliber.
Mf 900
Manufactured by Narishige
Sourced in Japan, United States
The MF-900 is a micromanipulator produced by Narishige. It is a precision instrument designed for the accurate positioning and control of microscopic objects or electrodes during various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pc 10, by Narishige (7 mentions)
P 97 microelectrode puller, by Narishige (3 mentions)
Axopatch 200b amplifier, by Molecular Devices (3 mentions)
Te300, by Nikon (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
P 1000, by Sutter Instruments (2 mentions)
Qcapture pro 6, by Olympus (2 mentions)
Pc 100, by Narishige (2 mentions)
Femtojet, by Narishige (2 mentions)
Eg 400, by Narishige (2 mentions)
Origin 7, by OriginLab (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bf150 110 10, by Sutter Instruments (1 mentions)
Nifedipine, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Zyla 4.2 cl10, by Oxford Instruments (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Pn 31, by Narishige (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Um 3c, by Narishige (1 mentions)
34 protocols using mf 900
1
Fabrication of AVIFJ Nozzle
2
Patch-Clamp Techniques for Action Potentials and Calcium Currents
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Standard patch-clamp techniques were used to record action potentials, and L-type calcium currents. Composition of intracellular fluid and extracellular fluid were shown in Table 7 and Table 8 , respectively. The signal was amplified using an HEKA EPC-10 patch-clamp amplifier (HEKA, Electronics, Ludwigshafen, Germany) and low-pass filtered at 5 kHz. Patch pipettes were fabricated from glass capillaries using a Sutter P-97 microelectrode puller (Novato, CA, USA) and the tips were heat polished with a microforge (NARISHIGE MF-900) to gain a resistance of 2–5 MΩ. Data acquisition was achieved using HEKA Patchmaster (V2x73.2). Data analysis and fit were performed using Clamfit and Graphpad Prism 6.0. The experiment was conducted at 37 °C. One cell can test one or more drugs, or multiple concentrations of the same drug but needs to be rinsed with extracellular fluid between each test. Nifedipine (Sigma) was used as a positive control to ensure cell quality. External solution containing 0.1% DMSO was applied as vehicle to establish the baseline. Recording was done at a temperature-controlled room of 35–37 degrees Celsius.
3
Micropipette Aspiration of Microbubbles
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Micropipettes were prepared by pulling borosilicate capillaries (WPI, 1 mm/0.5 mm outer/inner diameter) using a puller (PN-31, Narishige). Afterward, the micropipettes were sized to a few micrometers in diameter and bent using a microforge (MF-900, Narishige) to introduce the pipette horizontally in the observation chamber. The pipette was connected through tubing to a water tank attached to the piezoelectric pressure controller mentioned previously and then filled with water. The observation chamber was made of two glass coverslips separated with a few layers of parafilm and was filled with the solution containing the bubbles. The micropipette was brought into contact with a GGV (HFBI-coated microbubble) and a negative pressure was applied, resulting in the suction of the microbubble with the formation of a tongue of length L(t). The experiments were performed at room temperature (T = 23 ± 1 °C), and aspirated microbubbles were visualized with an inverted microscope (Nikon Eclipse Ti). Bright-field images were recorded with a sCMOS camera (Zyla-4.2-CL10, Andor) at a time interval between 0.2 and 1 s and operated using μManager (2.0 beta) open-source microscopy software (SI Appendix, Table S1 and S5 ).
4
Stabilizing Zebrafish Embryos for Micromanipulation
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Since our organism of interest is immersed in an aqueous non-viscous solution, oscillations of the zebrafish embryo are a major concern during micromanipulation. Thus, special care should be taken to assure a rigid stable connection between the embryo tissue and the tip of the micropipette. To achieve this a holding pipette (typically positioned on the left side of the preparation), immobilized the specimen while an opposing injection micropipette (on the right side) was introduced into the embryo tail.
For forging holding pipettes, thin wall borosilicate capillaries (inner diameter 0.75 mm, outer diameter 1.00 mm, length 10 mm, without inner filament) were used. Ideal holding pipettes have a flat straight break at the tip with large inner and outer diameter to provide better attachment and support to the zebrafish embryo. Fire-polishing was then performed to create a smooth surface to interface with the embryo tissue without damaging it, and produce an inner diameter (around 0.50 mm) best suited to hold the embryo as described in Brown and Flaming, 1974 (link), where a common Bunsen burner or a micro forge (MF-900, Narishige) were used to fire-polish the holding pipette.
For forging holding pipettes, thin wall borosilicate capillaries (inner diameter 0.75 mm, outer diameter 1.00 mm, length 10 mm, without inner filament) were used. Ideal holding pipettes have a flat straight break at the tip with large inner and outer diameter to provide better attachment and support to the zebrafish embryo. Fire-polishing was then performed to create a smooth surface to interface with the embryo tissue without damaging it, and produce an inner diameter (around 0.50 mm) best suited to hold the embryo as described in Brown and Flaming, 1974 (link), where a common Bunsen burner or a micro forge (MF-900, Narishige) were used to fire-polish the holding pipette.
5
Measuring Red Blood Cell Membrane Shear Modulus
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A borosilicate glass micropipette was used to aspirate the RBC membrane to determine membrane shear modulus through micropipette aspiration technique. Pipettes were drawn from borosilicate glass tubing (Sutter Instrument Model P-2000) and cut (Narishige MF-900) prior to mounting to the micromanipulator. The micropipette’s inner diameter was approximately 1 ± 0.25 µm. A pressure-drop rate of 1 Pa/s and a total pressure drop of 100 Pa were applied to aspirate and deform each cell. For each treated sample and control sample, a total of 20 cells were measured and analyzed accordingly. The aspiration was visualized on an Olympus IX71 microscope and processed by QCapture Pro 6.0. The maximum time taken for measurement of each sample was approximately 2 hours before replenishing with fresh sample. The recorded aspiration values were manually extracted and the shear modulus was calculated using the Hochmuth model22 (link).
6
Forming Planar Lipid Bilayers with Nanopores
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The contact
bubble bilayer (CBB) method33 (link),34 (link) was performed under
an inverted microscope (IX71; Olympus, Tokyo,
Japan), and images were recorded using a digital camera (MS-200; Bio
Craft, Tokyo, Japan). Glass pipettes for bubble formation and perfusion
inside the bubble were fabricated by pulling a borosilicate glass
capillary (BF100-50-10; OD/ID; 1.0/0.5 mm, Sutter Instrument, Novato,
CA) with a micropipette puller (PC-100; Narishige, Tokyo, Japan).
Then, the tips of the pipettes were cut to obtain a tip diameter of
around 30 μm and lightly polished using a micro-forge (MF-900;
Narishige). First, 10 mg/mL DPhPC solution (in n-decane)
(100 μL) was put on a slide glass. Next, two glass pipettes,
one filled with the buffer solution with 100 nM αHL and the
other filled with the buffer solution containing 100 nM target DNA
were immersed into the DPhPC solution. Then, two water bubbles were
formed from the pipettes by applying pressure inside the pipettes.
Finally, the pBLM was formed by manipulating the pipettes so the two
water bubbles came into contact. The pipette position was controlled
by micromanipulators (UM-3C; Narishige) under the microscope. The
pressure in the pipette was regulated by a micro injector (IM-9B;
Narishige). Ag/AgCl electrodes were placed in both glass pipettes.
The ionic current was measured by applying a transmembrane potential
of +180 mV.
bubble bilayer (CBB) method33 (link),34 (link) was performed under
an inverted microscope (IX71; Olympus, Tokyo,
Japan), and images were recorded using a digital camera (MS-200; Bio
Craft, Tokyo, Japan). Glass pipettes for bubble formation and perfusion
inside the bubble were fabricated by pulling a borosilicate glass
capillary (BF100-50-10; OD/ID; 1.0/0.5 mm, Sutter Instrument, Novato,
CA) with a micropipette puller (PC-100; Narishige, Tokyo, Japan).
Then, the tips of the pipettes were cut to obtain a tip diameter of
around 30 μm and lightly polished using a micro-forge (MF-900;
Narishige). First, 10 mg/mL DPhPC solution (in n-decane)
(100 μL) was put on a slide glass. Next, two glass pipettes,
one filled with the buffer solution with 100 nM αHL and the
other filled with the buffer solution containing 100 nM target DNA
were immersed into the DPhPC solution. Then, two water bubbles were
formed from the pipettes by applying pressure inside the pipettes.
Finally, the pBLM was formed by manipulating the pipettes so the two
water bubbles came into contact. The pipette position was controlled
by micromanipulators (UM-3C; Narishige) under the microscope. The
pressure in the pipette was regulated by a micro injector (IM-9B;
Narishige). Ag/AgCl electrodes were placed in both glass pipettes.
The ionic current was measured by applying a transmembrane potential
of +180 mV.
7
Precise Poking Probe Manipulation
8
In vitro Transcription and Microinjection of Ezrin-GFP mRNA
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In vitro transcription of Ezrin-GFP mRNA from pRN3-Ezrin-GFP plasmid was performed using mMessage mMachine transcription kit (AM1348) as described in Korotkevich et al. (2017) (link).
Microinjection was performed with an injector (Epperndorf, FemtoJet) and micromanipulators (Narishige, MON202-D) mounted on inverted epifluorescence microscope (Zeiss, Axio Observer.Z1). The incubation chamber on the microscope was kept at 33.5°C during microinjection. Injection needles were made by pulling capillaries (Warner Instruments, GC100TF-15) using a needle puller (Sutter Instrument, P-97) and bending their tips with a microforge (Narishige, MF-900). mRNAs were injected to the cytoplasm of zygotes at 22 h post-hCG, which were kept in a drop of 10 μl of H-KSOMaa covered with mineral oil. Before injection, RNA solution was centrifuged with 5,000g for 15 min at 4°C.
Microinjection was performed with an injector (Epperndorf, FemtoJet) and micromanipulators (Narishige, MON202-D) mounted on inverted epifluorescence microscope (Zeiss, Axio Observer.Z1). The incubation chamber on the microscope was kept at 33.5°C during microinjection. Injection needles were made by pulling capillaries (Warner Instruments, GC100TF-15) using a needle puller (Sutter Instrument, P-97) and bending their tips with a microforge (Narishige, MF-900). mRNAs were injected to the cytoplasm of zygotes at 22 h post-hCG, which were kept in a drop of 10 μl of H-KSOMaa covered with mineral oil. Before injection, RNA solution was centrifuged with 5,000g for 15 min at 4°C.
9
Whole-cell Recordings of Transfected HEK293 Cells
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Whole-cell recordings of transfected HEK293 cells were obtained at room temperature (25°C) using an Axopatch 200B amplifier (Axon Instruments). Electrodes were pulled with borosilicate glass capillaries by a programmable puller (P-1000, Sutter Instruments, USA) and heat-polished by a microforge (MF-900, Narishige, Japan), resulting in 1–3 MΩ resistances, before series resistance compensation of 70% or more. The internal solutions contained, (in mM): CsMeSO3, 135; CsCl2, 5; MgCl2, 1; MgATP, 4; HEPES, 5; and EGTA, 5; at 290 mOsm adjusted with glucose and at pH 7.3 adjusted with CsOH. The bath solution contained (in mM): TEA-MeSO3, 140; HEPES, 10; BaCl2, 10; 300 mOsm, adjusted with glucose and at pH 7.3 adjusted with TEAOH, all according to the previous report.24 (link)
10
Fabrication of Monodisperse Double-Emulsion Drops
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A classic glass-capillary microfluidic device was used to fabricate the W/O/W double-emulsion drops, as described previously [17 (link),45 (link)]. Briefly, two circular capillary tubes (ID of 0.58 mm, OD of 1.03 mm, World Pricision Instrument Inc., Sarasota, FL, USA) were given tapered openings of 38 and 170 μm in diameter using a micropipette puller (P-97, Sutter Instrument Inc., Novato, CA, USA) and a microforge (Narishige MF-900, Tokyo, Japan). The outside of the glass capillary tube for the inner fluid was hydrophobically functionalized with OTS to enhance the wettability of the capillary tube with oil phase, facilitating the fabrication of W/O/W double-emulsion drops. The two tapered circular capillary tubes were coaxially aligned and opposed to each other within a square glass capillary (ID of 1.05 mm), which were spaced from each other by 80 μm, as shown the optical microscope image in Figure 2 a. All the capillaries and needles used for channels were connected using a transparent epoxy. Such a configuration possessed the hydrodynamic flow-focusing and coflowing functions for one-step fabrication of highly monodisperse W/O/W double-emulsion drops.
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