The microinjection workstation consisted of an Olympus IX73 (Olympus, Japan) differential interference microscope with a UPlanFL N objective 4×, a UPlanFL N objective 10×, a LUCPlan FL N objective 40×, and a UPlanFL N objective 60× . In addition, the microscope was equipped with a TransferMan 4r micromanipulator set (Eppendorf, Germany) with a FemtoJet 4i microinjector (Eppendorf, Germany).
Transferman 4r
Manufactured by Eppendorf
Sourced in Germany
The TransferMan 4r is a precision micromanipulator designed for delicate cell and sample handling in life science research applications. It offers accurate 3D control and positioning of micropipettes or other tools with micrometer precision.
Automatically generated - may contain errors
Lab products found in correlation
30 0017 gc100 15b, by Harvard Apparatus (2 mentions)
Embryoscope, by Olympus (2 mentions)
Femtojet 4i, by Eppendorf (2 mentions)
M2 medium, by Merck Group (2 mentions)
Celltram vario, by Eppendorf (2 mentions)
Axio vert a1, by Zeiss (2 mentions)
Cd45 pe, by BioLegend (1 mentions)
Epcam, by Cell Signaling Technology (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Ovoil, by Vitrolife (1 mentions)
G gamete, by Vitrolife (1 mentions)
G 1 plus, by Vitrolife (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Female icr mice, by Charles River Laboratories (1 mentions)
Injectman 4, by Eppendorf (1 mentions)
Celltram air, by Eppendorf (1 mentions)
T7 high yield transcription kit, by Vazyme (1 mentions)
Embryo tested mineral oil, by Merck Group (1 mentions)
Cellsdirect one step qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using transferman 4r
1
Microinjection Workstation Setup
2
Cell Compression Assay Using Micromanipulator
3
Isolation and Characterization of CTC Clusters
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The captured, alive CTC clusters were stained with Alexa 488-conjugated antibodies against EpCAM (Cell Signaling Technology – Cat No: 5198S, (1:400)), prostate-specific membrane antigen (PSMA) (Biolegend – Cat No: 342506, Clone: LNI-17, (1:80)), and the contaminating WBCs were stained with PE-CD45 (TRITC) (BioLegend – Cat No: 368510, Clone: 2D1, (1:40)). The device with captured clusters was mounted in a Petri dish, and then imaged using inverted fluorescence microscope (Eclipse Ti, Nikon, Melville, NY). Identified clusters were directly micromanipulated from the device and transferred to the PCR tubes containing RLT (Qiagen) + BME (Sigma-Aldrich) buffer using Eppendorf TransferMan 4r micromanipulator. During micromanipulation, the dish was supplemented with 1× PBS to prevent microwells from drying, which was observed to take ~10 min at room temperature, Tubes were vortexed for 1 min and transferred to −80 °C freezer. Most of contaminating WBCs were observed to remain adhered to the device during cluster retrieval. Nevertheless, CTC clusters micromanipulated from Cluster-Wells were discharged into an empty Petri dish and were then repicked to ensure against potential artifacts from WBC contamination in RNA sequencing.
4
Imaging Techniques Using Zeiss Microscope
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Imaging was done using a Carl Zeiss LSM 800 microscope and a 63× objective. Glass coverslips were obtained from Menzel Glaser (24 × 36 mm, no. 0). Micropipettes were made from capillaries (1.0 OD × 0.58 ID × 150 L mm 30–0017 GC100-15b), purchased from Harvard Apparatus; they were stretched by using a micropipette puller from Sutter Instrument (model P-2000). Micromanipulators TransferMan 4r were purchased from Eppendorf. The pressure transducer used was DP103, provided by Validyne Engineering Corp. The pendent droplet tensiometer apparatus was from Teclis Instruments.
5
Intracytoplasmic Sperm Injection Protocol
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Microinjections were performed at ×400 magnification on a 37 °C heated stage inverted Nikon Eclipse Ti2 (Nikon, Tokyo, Japan). A Petri dish containing a microdroplet of ICSI ™ media in the center (ICSI ™, VitroLife, Sweden) under paraffin oil (OVOIL, VitroLife, Sweden) was used for sperms selection and immobilization. On the same dish, a microdroplet of G-Gamete ™ culture medium (G-Gamete ™, Vitrolife, Sweden) was used for placing the oocytes for microinjection. A single sperm was mechanically immobilized using the tip of the microinjection needle (Origio, USA) and then was aspirated inside the needle. The oocyte was held in place using a 35-degree angle holding micropipette (Origio, USA) with the polar body in the 6 or 12 o'clock position. Injection of a single spermatozoon within the oocyte cytoplasm was performed by using a micromanipulator (TransferMan® 4r, eppendorf, Germany). After ICSI, injected oocytes were cultured in G1-Plus ™ medium (G1-Plus ™, VitroLife, Sweden). Fertilization was confirmed by the presence of two pronuclei and the extrusion of the second polar body approximately 16–18 h after ICSI. The Fertilization Rate was calculated by dividing the number of obtained zygotes (2 PN) by the number of injected oocytes.
6
Blastocyst Injection for Trophoblast Stem-Like Cells
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Trophoblast stem-like cells (TSLCs) that expressed green fluorescent protein (GFP) were isolated and kept at a temperature of 4 °C. Afterward, a microinjection system (TransferMan-4r, Eppendorf) was used to inject 6–8 individual TSLCs into the cavity of a blastocyst at embryonic day 3.5. The embryos were then cultured in a G-2 medium for an additional 12 h before being transferred into the uterus of recipient animals. To assess the occurrence of successful implantations, the conceptus was gently exfoliated in PBS 10 days post-embryo transfer.
7
Trophectoderm Biopsy for PGT-A Analysis
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Embryo biopsy was performed immediately after the embryos were taken from the Embryoscope® using an OLYMPUS IX73 inverted microscope, a LIKOS (Hamilton Thorne) laser, TransferMan 4r (Eppendorf) micromanipulators, and HOLDING MPH-MED-30 (Origio) and BIOPSY MBB-FP-M-30 (Origio) micropipettes. Laser power was set on Validation mode (100% power - pulses of 430 microseconds) and no more than four laser shots were used to separate trophectoderm cells. After biopsy, tubing was performed according to the recommended protocol from the genetics laboratory (Genomics Perú). The embryos were kept vitrified until the results from PGT-A analysis were available.
8
Microinjection of Tubulin cRNAs in Mouse Oocytes
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Female ICR mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co. GV oocytes were isolated from the ovaries of Female ICR mice 44–52 h following injection with 10 IU pregnant mare serum gonadotropin (ShuSheng). Fully grown GV oocytes were collected and cultured in M2 medium (M7167, Sigma-Aldrich) with milrinone (2.5 μM) to maintain arrest at prophase. Microinjection was performed using a Leica Hoffman microscope (DMi8, Leica) equipped with a TransferMan 4r micromanipulator, InjectMan 4, and FemtoJet 4i (Eppendorf). About 0.1%–0.3% volumes of cRNA solution were microinjected into the cytoplasm of each mouse GV oocyte. Different cRNA concentrations were as follows: 5’FLAG-TUBB8 (200 ng/μL), 5’FLAG-TUBB4A (200 ng/μL), 5’FLAG-TUBB5 (200 ng/μL), 5’FLAG-TUBA1C (200 ng/μL), 5’FLAG-TUBB8△C-tail (200 ng/μL), 5’FLAG-TUBB8D435E (200 ng/μL), and 5’FLAG-KIF11 (500 ng/μL). Water was microinjected as a vehicle control. The injected GV oocytes were released into fresh M2 medium and further cultured on a 37 °C heating block in vitro. Oocytes were collected for subsequent analyses at indicated time points following culture.
9
Microinjection Workstation Setup
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The microinjection workstation consisted of an Olympus IX73 (Olympus, Japan) differential interference microscope with a UPlanFL N objective (4×), a UPlanFL N objective (10×) and an LUCPlan FL N objective (40×). The microscope was equipped with a TransferMan 4r (Eppendorf, Germany) micromanipulator set with the manual microinjectors CellTram Air and CellTram vario (Eppendorf, Germany).
10
Trophectoderm Biopsy for PGT-A Analysis
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Embryo biopsy was performed immediately after the embryos were taken from the Embryoscope® using an OLYMPUS IX73 inverted microscope, a LIKOS (Hamilton Thorne) laser, TransferMan 4r (Eppendorf) micromanipulators, and HOLDING MPH-MED-30 (Origio) and BIOPSY MBB-FP-M-30 (Origio) micropipettes. Laser power was set on Validation mode (100% power - pulses of 430 microseconds) and no more than four laser shots were used to separate trophectoderm cells. After biopsy, tubing was performed according to the recommended protocol from the genetics laboratory (Genomics Perú). The embryos were kept vitrified until the results from PGT-A analysis were available.
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