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Microloader

Manufactured by Eppendorf
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The Eppendorf Microloader is a precision pipetting tool designed for the accurate transfer of small liquid volumes. It features a narrow tip to facilitate the precise aspiration and dispensing of liquids in the microliter range.

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Lab products found in correlation

Hyaluronidase, by Merck Group (2 mentions) Femtotip 2, by Eppendorf (2 mentions) Femtojet microinjector, by Narishige (2 mentions) Nt 88ne, by Narishige (2 mentions) M2 medium, by Merck Group (2 mentions) Femtotip, by Eppendorf (2 mentions) Dextran cascade blue, by Thermo Fisher Scientific (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) Glass micropipette, by Sutter Instruments (2 mentions) Nih 3t3 fibroblasts, by American Type Culture Collection (2 mentions) Femtojet 4i microinjector, by Eppendorf (1 mentions) Tricaine, by Merck Group (1 mentions) Seahorse xf96 cell culture plate, by Agilent Technologies (1 mentions) Cytochalasin b, by Merck Group (1 mentions) P 2000 puller, by Sutter Instruments (1 mentions) Femtojet 4i, by Eppendorf (1 mentions) C11440, by Hamamatsu Photonics (1 mentions) Tcm 199, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Microloader tip (11 citations) Microloader pipette tip (5 citations) Shortened microloader tip (2 citations)

19 protocols using microloader

1

siRNA Microinjection for Zygote Modification

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All siRNAs used in this study were purchased from RiboBio. The scrambled siRNA and Trim21 mRNA was injected into the zygotes as the negative control. We placed zygote-stage embryos in 150 µg/mL hyaluronidase (4272; Sigma) to digest the outer granule cells. The siRNA was centrifuged at 12,000 rpm for 10 min at 4 °C, and stored at 4 °C until use. Then, microinjection was performed using a FemtoJet 4i microinjector (Eppendorf, Hamburg, Germany) and E LIPSE Ti micromanipulators (Nikon, Tokyo, Japan). For injection, a glass capillary Femtotip (Eppendorf) was loaded with 2 μL of RNA mixtures using a Microloader (Eppendorf), and the solution was injected into the cytoplasm in a drop of M2 medium (M7167; Merck). The injection volume was approximately 2–5 pL. The injection conditions consisted of an injection pressure of 250 hPa, compensation pressure of 60 hPa, and injection time of 0.7 s. Immediately after microinjection, embryos were cultured in KSOM medium at 37 °C in 5% CO2.
Li J., Zhang J., Hou W., Yang X., Liu X., Zhang Y., Gao M., Zong M., Dong Z., Liu Z., Shen J., Cong W., Ding C., Gao S., Huang G, & Kong Q. (2022). Metabolic control of histone acetylation for precise and timely regulation of minor ZGA in early mammalian embryos. Cell Discovery, 8, 96.
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2

Calsequestrin Protein Soaking for Anomalous Signal

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We initially attempted to identify calcium sites using anomalous signal from calcium (CaCl2) added to the crystallization condition described above. We were unsuccessful, likely due to a combination of several factors. The calcium absorption edge is unreachable with conventional tunable x-ray sources and in normal atmosphere; thus, it can only be approached, with resulting weakened anomalous signal. In addition, calsequestrin has an average Kd for calcium of 1 mM. Thus, occupancy at a typical site would be expected to be lower as compared to other calcium-binding proteins. Presence of sulfate in the crystallization condition limited calcium concentrations to approximately 14 mM and below, above which a precipitate was observed. Although this limit is above the Kd, it was insufficient for robust anomalous signal. Crystals of calsequestrin that formed in trace calcium were therefore soaked in YbCl3. Hanging drops containing calsequestrin crystals were uncovered and an Eppendorf Microloader was used to inject 2 μL crystallization drops (1 μL protein and 1 μL mother liquor) with 200 μL of 2 M YbCl3. Data were collected within 5 minutes with no back-soaking.
Titus E.W., Deiter F.H., Shi C., Wojciak J., Scheinman M., Jura N, & Deo R.C. (2020). The structure of a calsequestrin filament reveals mechanisms of familial arrhythmia. Nature structural & molecular biology, 27(12), 1142-1151.
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3

Stabilizing Seahorse Assay Embryos

Cited 1 time
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Tricaine (E10521, Merck, United States) was used to reduce embryo movement in the Seahorse well and to stabilize OCR, as previously shown by Raftery et al. (2017) (link). The embryos were incubated in a petri dish containing 125mg/ml Tricaine in E3 medium and then transferred into the Seahorse XF96 cell culture plates (101,085, Agilent, United States) with a 100μl cut pipet tip before adding 170μl of XF base medium with minimal DMEM (103,334, Agilent, United States) to each well. The central position of the embryos was checked with a microscope and corrected with a shortened Microloader™ pipet tip (EP5242956003, Eppendorf, Germany), if necessary. For analyzing the effect of sedation on the variability of OCR, Tricaine was injected via port A of the XF96 cartridge.
Rollwitz E, & Jastroch M. (2021). Plate-Based Respirometry to Assess Thermal Sensitivity of Zebrafish Embryo Bioenergetics in situ. Frontiers in Physiology, 12, 746367.
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4

Ago2 Knockdown via siRNA Microinjection

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All small interfering RNAs (siRNAs) were purchased from GenePharma. Ago2-siRNAs was designed to specifically target Ago2 and on the basis of 30%-52% GC content and avoiding of internal repeats (5′–3′). Ago2-siRNA1: GCAAAGAUCGCAUCUUUAATT; Ago2-siRNA2: GCCAGUGAUCGAGUUUGUUTT; Ago2-siRNA3: GCAGAAACACACCUACCUUTT; the scrambled siRNA used as negative control. All siRNAs were modified with 2′Fluoro rU/C to increase their annealing temperature. The prediction of off-target effects was described in result. To perform microinjection, zygote-stage embryos were placed in 150 µg/mL hyaluronidase (Sigma) to digest the outer granule cells. SiRNAs was centrifuged at 12,000 rpm for 10 minutes at 4 °C and placed at 4 °C for use. Then, siRNA microinjection was carried out with an Eppendorf FemtoJet microinjector and Narishige NT-88NE micromanipulators. For injection, a glass capillary Femtotip II (Eppendorf) was loaded with 2 μL of 10 µM siRNA by a microloader (Eppendorf), and the solution was injected into the cytoplasm in a 100 μL drop of M2 medium (Sigma) plus 5 μg/mL cytochalasin B (Sigma). The injection volume was approximately 2–5 pL. The injection conditions consisted of 250 hPa injection pressure, 60 hPa compensation pressure and 0.7 s injection time. Immediately after microinjection, embryos were cultured in KSOM medium at 37 °C in 5% CO2.
Zhang J.M., Hou W.B., Du J.W., Zong M., Zheng K.L., Wang W.J., Wang J.Q., Zhang H., Mu Y.S., Yin Z., Ding C.M., Sun Q.Y., Liu Z.H, & Kong Q.R. (2020). Argonaute 2 is a key regulator of maternal mRNA degradation in mouse early embryos. Cell Death Discovery, 6, 133.
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5

Fabrication of Nanopipettes and Microinjection Pipettes

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To fabricate nanopipettes, borosilicate glass filaments (outer diameter 1.00 mm, inner diameter 0.58 mm) were pulled to an outer diameter of approx. 100 nm. The P2000 puller (Sutter Instruments) used the following program:
Line1: HEAT:350, FIL:3, VEL:30, DEL:220, PULL:0
Line2: HEAT:330, FIL:2, VEL:27, DEL:180, PULL:250
For comparison, microinjection pipettes (Femtotips, Eppendorf) with an inner diameter of 0.5 μm were purchased. Both pipette types were filled from the back with a microloader (Eppendorf).
Simonis M., Hübner W., Wilking A., Huser T, & Hennig S. (2017). Survival rate of eukaryotic cells following electrophoretic nanoinjection. Scientific Reports, 7, 41277.
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6

Nanopipette-Mediated Cell Transfection System

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Infinitesimal’s point-click-transfect NFP-E system was employed in all experiments. Glass nanopipette tips (Eppendorf) with an inside diameter of 500 nm (Figure 1b) were employed in the transfection experiments. Nanopipette tips were first loaded with the desired solution using a micro-loader (Eppendorf) and subsequently mounted on Infinitesimal’s NFP-E system with XYZ motion control achieved via three linear piezo actuators (closed loop, resolution < 10 nm). Infinitesimal’s software and electronic circuit were used to measure the resistance across the nanopipette tip (150 Hz sampling rate) and to automatically apply bi-level electroporation pulses. The movement of the nanopipette tip was observed under an inverted microscope (Nikon Eclipse) coupled to a CCD camera (Andor Zyla). When the resistance between the tip and the cell increased by approximately 1%, bilevel electroporation pulses (15 V 0.5 ms, 10 V 2.5 ms typical; Figure S2, Supplementary Information) were applied at a frequency of 50 Hz. Each train consisted of 50 such bilevel pulses, and typically 1 or 2 trains were used in the delivery experiments.
Nathamgari S.S., Pathak N., Lemaitre V., Mukherjee P., Muldoon J.J., Peng C.Y., McGuire T., Leonard J.N., Kessler J.A, & Espinosa H.D. (2020). Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Post-Pulse Electro-pore Dynamics. Small (Weinheim an der Bergstrasse, Germany), 16(43), e2002616.
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7

Microinjection System for Live Cell Imaging

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The microinjection system included a microinjector (FemtoJet 4i, Eppendorf) and a micro-manipulator (MS314, WPI) at a step resolution of 0.5 μm. Injected liquid was loaded with a micro-loader (Eppendorf) and then injected into the cells. All the injection process was observed under a charge-coupled device camera (C11440, Hamamatsu), which was a part of the Nikon eclipse Ti-based N-STORM microscopy system.
Ma Y., Han X., de Castro R.B., Zhang P., Zhang K., Hu Z, & Qin L. (2018). Analysis of the bystander effect in cone photoreceptors via a guided neural network platform. Science Advances, 4(5), eaas9274.
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8

Nanopore Functionalization Protocol

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The Na‐probe was firstly mixed with TCEP and incubated for 30 min. Then 10 µM activated Na‐probe was added into one nanopore by an Eppendorf Microloader for 16 h reaction with Au layer on the interior surface.[9b] The same modification step was employed for establishing the K‐nanopore. Afterward, the θ‐nanopipette was backfilled and immersed with 1 mM MCH for 10 min. Note that the θ‐nanopipette was rinsed repeatedly with the buffer solution between the modification steps.
Shi X., Liu F., Wang B., Yu S., Xu Y., Zhao W., Jiang D., Chen H, & Xu J. (2022). Functional nucleic acid engineered double‐barreled nanopores for measuring sodium to potassium ratio at single‐cell level. Exploration, 2(5), 20220025.
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9

Calsequestrin Protein Soaking for Anomalous Signal

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We initially attempted to identify calcium sites using anomalous signal from calcium (CaCl2) added to the crystallization condition described above. We were unsuccessful, likely due to a combination of several factors. The calcium absorption edge is unreachable with conventional tunable x-ray sources and in normal atmosphere; thus, it can only be approached, with resulting weakened anomalous signal. In addition, calsequestrin has an average Kd for calcium of 1 mM. Thus, occupancy at a typical site would be expected to be lower as compared to other calcium-binding proteins. Presence of sulfate in the crystallization condition limited calcium concentrations to approximately 14 mM and below, above which a precipitate was observed. Although this limit is above the Kd, it was insufficient for robust anomalous signal. Crystals of calsequestrin that formed in trace calcium were therefore soaked in YbCl3. Hanging drops containing calsequestrin crystals were uncovered and an Eppendorf Microloader was used to inject 2 μL crystallization drops (1 μL protein and 1 μL mother liquor) with 200 μL of 2 M YbCl3. Data were collected within 5 minutes with no back-soaking.
Titus E.W., Deiter F.H., Shi C., Wojciak J., Scheinman M., Jura N, & Deo R.C. (2020). The structure of a calsequestrin filament reveals mechanisms of familial arrhythmia. Nature structural & molecular biology, 27(12), 1142-1151.
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10

Knockdown of Key RNA Regulators in Oocytes

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To perform DICER1, DROSHA and DGCR8 knockdown experiments, the granulosa cells of oocytes at germinal vesicle (GV) stage were denuded. Then locked nucleic acid (LNA)-siRNA microinjections were carried out with an Eppendorf FemtoJet microinjector and Narishige NT-88NE micromanipulators. For injection, a glass capillary Femtotip II (Eppendorf) was loaded with 10 pL of 10 mM LNA-DICER1, LNA-DROSHA or LNA-DGCR8 (Exiqon) by microloader (Eppendorf) and the solution was injected into the cytoplasm of GV oocytes in a 200-mL drop of manipulation medium (TCM-199 (Invitrogen) plus 30 mg mL À1 bovine serum albumin (BSA)) supplemented with 7.5 mg mL À1 cytochalasin B. The injection conditions consisted of 250 hPa injection pressure, 60 hPa comensation pressure and 0.7 s injection time. Immediately after microinjection, oocytes were washed and co-cultured with mural granulosa cells in maturation medium. A scrambled LNA-siRNA (Exiqon) was used as a negative control (NC; Wei et al. 2011) (link).
Liu W., Zhao Q., Piao S., Wang C., Kong Q, & An T. (2017). Endo-siRNA deficiency results in oocyte maturation failure and apoptosis in porcine oocytes. Reproduction, fertility, and development, 29(11).
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