Glass micropipette

Manufactured by Sutter Instruments

Sourced in United States

Glass micropipettes are precision laboratory tools used for transferring small volumes of liquids. They are typically made of borosilicate glass and feature a narrow, tapered tip that allows for the accurate and controlled dispensing of microliter or nanoliter quantities of fluids.

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Lab products found in correlation

Dextran cascade blue, by Thermo Fisher Scientific (2 mentions) Microloader, by Eppendorf (2 mentions) Phenol red free mem, by Thermo Fisher Scientific (2 mentions) Anxv pacific blue, by Thermo Fisher Scientific (2 mentions) Rat basophilic cells rbl, by American Type Culture Collection (2 mentions) Nih 3t3 fibroblasts, by American Type Culture Collection (2 mentions) Adenosine, by Merck Group (1 mentions) Picospritzer 3, by Parker Hannifin (1 mentions) 40 m cell strainer, by BD (1 mentions) Glutamine, by Thermo Fisher Scientific (1 mentions) Neurobasal, by Thermo Fisher Scientific (1 mentions) P 1000, by Sutter Instruments (1 mentions) Multiclamp 700b amplifier, by Molecular Devices (1 mentions) Matlab, by MathWorks (1 mentions) Femtojet 5247, by Eppendorf (1 mentions) Pc 10 puller, by Narishige (1 mentions)

9 protocols using glass micropipette

Local Delivery of Adenosine and Inhibitors

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For local application of solutions into the rat brain, a glass micropipette (1.5 mm outside diameter, 0.86 mm inside diameter; Sutter Instruments, Novato, CA) was pulled and bumped to form an outer tip diameter of ~10 μM (P-97, Sutter Instruments). The micropipette was fixed to the shank of the MEA recording site using sticky wax, with the tip of the pipette secured ~300 μm above the most distal recording site. The micropipette was filled with a solution of either 200 μM adenosine (Sigma-Aldrich) or 200 μM dipyridamole (DPR) (Sigma-Aldrich), a competitive equilibrative nucleoside transporter (ENT) inhibitor that produces an increase in extracellular adenosine levels in vivo (Park and Gidday, 1990 (link)). All drugs were dissolved to their final concentrations in physiological saline, sterile filtered (0.20 μm), and adjusted to a pH of 7.4. The micropipette used for drug delivery was connected via tubing to a Picospritzer III (Parker-Hannifin, Cleveland, OH) with settings adjusted to consistently deliver volumes between 200 and 400 nL. Pressure was applied from 10–30 psi for 0.3–3 s and volume displacement was monitored via stereomicroscope.

Hinzman J.M., Gibson J.L., Tackla R.D., Costello M.S., Burmeister J.J., Quintero J.E., Gerhardt G.A, & Hartings J.A. (2015). Real-time monitoring of extracellular adenosine using enzyme-linked microelectrode arrays. Biosensors & bioelectronics, 74, 512-517.

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Primary Cortical Neuron Culture and Cloning

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Primary cortical cultures were prepared from postnatal mouse pups. As previously described^{37 (link)}, the cortex was cut into small pieces and digested at 37 °C for 15 min. To stop digestion, the complete medium (DMEM supplemented with 10% FBS) was applied. After digestion, the tissue suspension was filtered with a 40-µm cell strainer (Falcon) and centrifuged at 1000×g for 8 min. Cells were counted with cell counting equipment and seeded in 24-well plate at 37 °C with 5% CO₂. After 4-h incubation, the culture medium was removed and replaced with maintenance medium, containing Neurobasal (Gibco), B27 (Invitrogen), and glutamine (Invitrogen). Anti-Tuj-1 (1;1000 mouse, Beyotime, China) immunostaining was used for characterization of the cultured neurons. HEK293, U251, and SH-SY5Y cells were grown in DMEM supplemented with 10% FBS at 37 °C with 5% CO₂. For selecting the single clone cells, lentiviral infected cells were placed in 96-well plate by glass micropipette (Sutter Instrument). The genotypes of single clone cell lines were characterized by PCR and sanger sequencing.

Piao X., Meng D., Zhang X., Song Q., Lv H, & Jia Y. (2022). Dual-gRNA approach with limited off-target effect corrects C9ORF72 repeat expansion in vivo. Scientific Reports, 12, 5672.

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Lipid Membrane Probes and Cell Lines

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Synthetic lipids were obtained from Avanti. Di-4-ANEPPDHQ (Di4), BSA (fraction V), Phenol red-free MEM, AnxV-Pacific Blue, and dextran-cascade blue (3000 Da) were purchased from Thermo Fisher. NR12S was kindly provided by A. Klymchenko and Mikhail Bogdanov. All other chemicals were purchased from Sigma. Rat basophilic cells (RBL) and NIH 3T3 fibroblasts were purchased from ATCC. Glass micropipettes were from Sutter Instruments and microloaders from Eppendorf. For analysis of red blood cells, 6 channel IBIDI™ slides were used.

Lorent J., Levental K., Ganesan L., Rivera-Longsworth G., Sezgin E., Doktorova M., Lyman E, & Levental I. (2020). Plasma membranes are asymmetric in lipid unsaturation, packing, and protein shape. Nature chemical biology, 16(6), 644-652.

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Patch-Clamp Electrophysiology of Hippocampal Neurons

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The external solution for patch-clamp electrophysiology experiments consisted of (in mM): NaCl 125, D-glucose 10, NaHCO₃ 26, NaH₂PO₄ 1.25, KCl 4, CaCl₂ 0.25. This solution was equilibrated with a mixture of 95 vol% O₂ and 5 vol% CO₂ for at least 30 minutes with a resulting pH of about 7.4. This modified aCSF solution was chosen to promote neuronal excitability [14 (link)]. The internal solution consisted of (in mM): potassium-D-gluconate 130, EGTA 5, NaCl 4, CaCl₂ 0.5, HEPES 10, pH 7.3. Glass micropipettes (Sutter Instruments O.D. 1.5 mm) were pulled using a Sutter Instruments model P-1000 and fabricated to maintain an initial resistance of 3–4 MΩ. Neuronal membrane responses were recorded using a Multiclamp 700B amplifier (Molecular Devices, Foster City, CA). To assess excitability of CA1 hippocampal pyramidal neurons, we used a single-unit extracellular (loose) or cell-attach protocol with a relatively low membrane resistance (<100 MΩ) at the holding membrane potential of 0 mV [18 ]. Voltage current commands and digitization of the resulting voltages and currents were performed with Clampex 8.3 software (Molecular Devices, Foster City, CA) running on a compatible computer. Resulting current traces were analyzed offline using MiniAnalysis software (Synaptosoft, Inc., Decatur, GA).

Raol Y.H., Joksimovic S.M., Sampath D., Matter B.A., Lam P.M., Kompella U.B., Todorovic S.M, & González M.I. (2020). The role of KCC2 in hyperexcitability of the neonatal brain. Neuroscience letters, 738, 135324.

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Measuring Precision of Neuronal Spike Timing

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The extracellular activity was obtained with glass micropipettes filled with 2M NaCl (Sutter Instrument). The electrode was advanced into the left flocculus using a manipulator (Narishige International, Amityville, NY, USA). Signals were amplified and band-pass filtered (400–5000 Hz). The single-unit activity was recorded using a Plexon system at a sampling rate of 20 kHz for offline analysis. Raw spike signals were imported in MATLAB (The MathWorks, Natick, MA, USA) and analyzed. The spike timing precision was quantified using a coefficient of variation (CV). CV is calculated using the formula,

C V = \frac{σ I S I}{μ I S I}

, where σ and μ are the standard deviation and mean of the inter-spike intervals (ISI). In addition to CV and frequency of simple spikes, the CV2, which measures short-scale regularity in spike trains, was also calculated where

C V 2 = \frac{2 |I S I_{n + 1} - I S I_{n}|}{(I S I_{n + 1} - I S I_{n})}

Chang H.H., Cook A.A., Watt A.J, & Cullen K.E. (2022). Loss of Flocculus Purkinje Cell Firing Precision Leads to Impaired Gaze Stabilization in a Mouse Model of Spinocerebellar Ataxia Type 6 (SCA6). Cells, 11(17), 2739.

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CRISPR-Mediated Oocyte Microinjection

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Microinjection was performed on the heated stage of a Zeiss Axiovert-35 inverted microscope (Zeiss, Jena, Germany) fitted with Eppendorf micromanipulators and Femtojet 5247 injector (Eppendorf, Hauppauge, USA). Glass micropipettes with an outer diameter of 1.0 mm and an inner diameter of 0.78 mm were pulled to a fine point of <1.0 μm (Sutter Instrument, Navato, USA). The mixture of CRISPR RNA (crRNA, 100 ng/µl) and 20 ng/µl of gRNAs was microinjected into cytoplasm of oocyte (designated as Uba52gRNA group). crRNA (100 ng/μl) without gRNAs was injected as a control. Surviving oocytes were washed three times in fertilization medium and used for IVF.

Mao J., O'Gorman C., Sutovsky M., Zigo M., Wells K.D, & Sutovsky P. (2018). Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development. Biology Open, 7(10), bio035717.

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Lipid Membrane Probes and Cell Lines

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Whole-Cell Patch Clamp of TRPC Channels

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HEK293 cells stably expressing TRPC channels were seeded in 35 mm dishes at least 5 h before whole-cell patch clam recordings were performed. Recording pipettes were pulled from micropipette glass (Sutter Instrument) to 2–5 MΩ when filled with a pipet solution containing (in mM): 110 CsCl, 10 HEPES, 10 BAPTA, 1 MgCl₂, 4.77 CaCl₂, pH adjusted to 7.2 with HCl (400 nM free Ca²⁺) and placed in the bath solution containing (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4. Individual cells were voltage-clamped in the whole-cell mode using either an EPC9 or EPC10 (HEKA Instruments Inc., Bellmore, NY) amplifier. Voltage commands were made from the PatchMaster program (version 2.60; HEKA), and currents were recorded at 5 kHz. Voltage ramps of 100 ms to –100 mV after a brief (20 ms) step to +100 mV from holding potential of 0 mV were applied every 1 s. Cells were continuously perfused with the bath solution through a gravity-driven multichannel system with the desired channel opening placed about 50 μm away from the cell being recorded. For some recordings, CaCl₂ in the bath solution was reduced to 0.5 or 0.1 mM as indicated in the figure legends. Drugs were diluted in the bath solution with the desired Ca²⁺ concentration and applied to the cell through perfusion.

Qu C., Ding M., Zhu Y., Lu Y., Du J., Miller M., Tian J., Zhu J., Xu J., Wen M., Er-Bu A., Wang J., Xiao Y., Wu M., McManus O.B., Li M., Wu J., Luo H.R., Cao Z., Shen B., Wang H., Zhu M.X, & Hong X. (2017). Pyrazolopyrimidines as Potent Stimulators for Transient Receptor Potential Canonical 3/6/7 Channels. Journal of medicinal chemistry, 60(11), 4680-4692.

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Patch-clamp analysis of Drosophila neurons

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Approximately 3 h after plating, GFP-expressing dissociated primary Drosophila neurons were identified and selected for whole-cell patch clamp. Before measurement of resting membrane potential, the bath solution was replaced with room temperature HL-3 (70 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 20 mM MgCl₂, 10 mM NaHCO₃, 115 mM sucrose, 5 mM trehalose, and 5 mM Hepes; pH 7.2). The pipette solution used contained the following: 109 mM K-gluconate, 10 mM NaCl, 1.7 mM MgCl₂, 0.085 mM CaCl₂, 0.94 mM EGTA, 2 mM ATP, and 8.5 mM Hepes; pH 7.2. Recording pipettes were pulled from micropipette glass (Sutter Instruments) to 8 to 10 M-Ω on a PC-10 puller (Narishige). Clamping was performed with an EPC10 (HEKA Instruments) amplifier. Commands were made from the PatchMaster program (version 2 × 90.1; HEKA). G-Ω seal was achieved under voltage-clamping mode with V membrane holding at −70 mV to prevent cell excitation after the membrane was broken. Cells with resistance higher than 1 G-Ω were used for measuring resting membrane potential. Currents were clamped at zero, and voltage was continuously recorded at 10 kHz for a minimum of 3 min per cell.

Wong C.O., Karagas N.E., Jung J., Wang Q., Rousseau M.A., Chao Y., Insolera R., Soppina P., Collins C.A., Zhou Y., Hancock J.F., Zhu M.X, & Venkatachalam K. (2021). Regulation of longevity by depolarization-induced activation of PLC-β–IP3R signaling in neurons. Proceedings of the National Academy of Sciences of the United States of America, 118(16), e2004253118.

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