All animal experiments were approved by the Northern Stockholm Experimental Animal Ethical Committee. Zebrafish embryos were raised at 28°C under standard experimental conditions. Zebrafish embryos at the age of 24-hpf were incubated in water containing 0.2 mmol/L 1-phenyl-2-thio-urea (PTU, Sigma). At 48-hpf, zebrafish embryos were dechorionated with a pair of sharp-tip forceps and anesthetized with 0.04 mg/mL of tricaine (MS-222, Sigma). Anesthetized embryos were subjected for microinjection. CAL33 RR tumor cells were labeled in vitro with a Vybrant DiD cell-labeling solution (LifeTechnologies). Tumor cells were resuspended in PBS and approximatively 5 nL of the cell solution were injected into the perivitelline space (PVS) of each embryo by an Eppendorf microinjector (FemtoJet 5247). Non-filamentous borosilicate glass capillaries needles were used for injection and the injected zebrafish embryos were immediately transferred into PTU aquarium water. Fish are immediately treated with onvansertib (50 nM). For Figure 5 E-F, 24 h after injection, only zebrafish with metastasis were chosen and treated with onvansertib (50 nM). Zebrafish embryos were monitored 72 h for investigating tumor metastasis using a fluorescent microscope (Nikon Eclipse 90).
Femtojet 5247
Manufactured by Eppendorf
Sourced in Germany, United States
The FemtoJet 5247 is a microinjector device designed for precise liquid handling. It is capable of delivering ultra-small volume samples with high accuracy and repeatability. The core function of the FemtoJet 5247 is to provide controlled microinjection for a variety of applications in life science research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Ms 222, by Merck Group (6 mentions)
Vybrant did cell labeling solution, by Thermo Fisher Scientific (3 mentions)
Transferman nk2, by Eppendorf (3 mentions)
Szx16 microscope, by Olympus (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Szx16, by Olympus (1 mentions)
Bitplane, by Oxford Instruments (1 mentions)
Thunder imaging system, by Leica (1 mentions)
P 2000, by Sutter Instruments (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
B100 75 10, by Sutter Instruments (1 mentions)
T7 ribomax express rnai system, by Promega (1 mentions)
Lsm 700 system, by Zeiss (1 mentions)
Eclipse 90, by Nikon (1 mentions)
Phenylthiourea, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Eppendorf (1 mentions)
Celltracker cm dii dye, by Thermo Fisher Scientific (1 mentions)
Megashortscript kit, by Thermo Fisher Scientific (1 mentions)
T7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Puc57 sgrna expression vector, by Addgene (1 mentions)
20 protocols using femtojet 5247
1
Zebrafish Model of Tumor Metastasis
2
Ovary and Fat Body miRNA Analysis
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After eclosion, the 3-day-old females were anesthetized on ice and a total of 1 μL agomiR/antagomiR solution (20 μM) was injected into the ventral abdomen of female adults using an Eppendorf micromanipulation system (FemtoJet 5247; Eppendorf, Hamburg, Germany) as described previously [65 (link)]. The same volume of random shuffled sequence was used as a negative control (NC). At 24 h post injection, flies were sacrificed to dissect the ovary and fat body for RNA extraction and the subsequent qRT–PCR analysis (10 flies in each treatment group). The miRNA mimic (agomiR), inhibitors (antagomiR) and negative control (NC) oligonucleotides were purchased from GenePharma (Shanghai, China). The sequences are listed in Table S2.
3
Silkworm Embryo Microinjection Protocol
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A diapausing strain, Dazao, which is wildly used as a wild type silkworm, was utilized in this study. The larvae were reared with fresh mulberry leaves at 25°C, 75% RH. Parental embryos (P) were incubated at 15°C and 75% RH to produce nondiapusing eggs (G0), which are suitable for microinjection. The microinjections were performed utilizing TransferMan NK2 micromanipulator and Femto Jet 5247 microinjector (Eppendorf) under an SZX16 microscope (Olympus) as we reported previously [11 (link)]. For transient assays, embryos were injected within 5 hours after oviposition and harvested 3 days post injections. For germline assays such as heritable chromosomal deletion using TALEN-B3 and ssODN B3-794, embryos were injected within 2 hours after oviposition and allowed to develop to moths. Mosaic mutations were observed as early as the 3rd instar. The resulting G0 males were first crossed with G0 females and the remaining uncopulated G0 males were crossed with wild-type females. The G1 mutations were checked for the translucent skin phenotype on the 3rd instar, and all the positive mutations were allowed to develop to the moths.
4
Silkworm Embryo Microinjection Protocol
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Two B. mori strains, which were reared on fresh mulberry leaves, were used in our study. Nistari (N4) is a non-diapausing strain and the preparation of embryo for microinjection is as Tamura et al. described38 (link). Dazao is a diapausing strain and the embryos were prepared as Zhao et al. described39 . mRNA samples or piggyBac transgenic vectors were microinjected into silkworm embryos within 2 h after oviposition. Microinjections were performed using TransferMan NK2 micromanipulator and Femto Jet 5247 microinjector (Eppendorf) under a microscopy SZX16 (Olympus). The injection opening was sealed with instant glue (Konishi co.) and injected embryos were incubated at 25°C and 90%RH. Larvae hatched from the injected embryos were collected and reared on fresh mulberry leaves. The resulted moths were sibling crossed to screen the mutant.
5
Zebrafish Metastasis Assay with Cabozantinib
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All animal experiments were approved by the Northern Stockholm Experimental Animal Ethical Committee. Zebrafish embryos were raised at 28°C under standard experimental conditions. Zebrafish embryos at the age of 24 hpf were incubated in aquarium water containing 0.2 mmol/L 1-phenyl-2-thio-urea (PTU, Sigma). At 48-hpf, zebrafish embryos were dechorionated with a pair of sharp-tip forceps and anesthetized with 0.04 mg/mL of tricaine (MS-222, Sigma). Anesthetized embryos were subjected to microinjection. CAL33RR tumor cells were labeled with a Vybrant DiD cell-labeling solution (LifeTechnologies). Tumor cells were resuspended in PBS and approximately 5 nL of the cell solution was injected into the perivitelline space (PVS) of each embryo by an Eppendorf microinjector (FemtoJet 5247). Non-filamentous borosilicate glass capillarie needles were used for injection. The injected zebrafish embryos were transferred into PTU aquarium water. For Figure 6 , fish were treated with cabozantinib (1 μM). For Figure 7 , 24 h after injection, only zebrafish with metastases were chosen and treated with cabozantinib (1 μM). Zebrafish embryos were monitored for 72 h to investigate tumor metastasis using a fluorescent microscope (Nikon Eclipse 90).
6
Human NK Cell Engraftment in Zebrafish
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MEL270 cells that were pre-labelled with red fluorescent DIL (Thermo Fisher) and human NK cells were mixed at a 1:3 ratio (MEL270: NK). Zebrafish embryos at the age of 24 hpf (hours post fertilization) were incubated in water containing 0.2 mmol/L 1-phenyl-2-thio-urea (PTU, Sigma). At 48-hpf prior to microinjection, zebrafish embryos were dechorionated and anesthetized with 0.04 mg/mL of tricaine (MS-222, Sigma). The microinjection of human cell mixture was performed by infusing 5nL (approximately 500 cells in total) into the perivitelline space of each larvaes using an Eppendorf microinjector (FemtoJet 5247, Eppendorf and Manipulator MM33-Right, Märzhäuser Wetziar). Successfully injected larvae were transferred into PTU aquarium water at 33°C for 48 h incubation before fixation with 4% paraformaldehyde (PFA) for image acquisition. 3D Images of zebrafish larvaes were acquired on Thunder Imaging System (Leica Microsystems) under 4X objectives. Batch quantification of different treatment groups were done using IMARIS software. (Bitplane, Oxford Instruments).
7
Dye Microinjection into Perivitelline Space
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Needles used for dye microinjections were fabricated from borosilicate capillaries (B100-75-10, Sutter Instruments) using a pipette puller (P-2000, Sutter Instruments). Pipettes were backfilled with 25-fold diluted CellMask Deep Red Plasma Membrane stain (Thermo Fisher Scientific) using a gel loading tip (Eppendorf). Pipettes were broken at the tip by gently nudging the pointed end against an edge of a coverslip, under a drop of oil.
To inject dye, the tip of a pipette was carefully pushed against the vitelline membrane so as to insert it into the perivitelline space without piercing the cellular layer. An Eppendorf injector (FemtoJet 5247, Eppendorf) was used with the following settings: compensation pressure: 10 hPa, injection pressure: 220 hPa. Typically, the injection was performed using the ‘clean’ command such that maximal pressure was applied.
After injection, the embryos were cut along the side opposite to the site of injection for subsequent cantilever insertion.
To inject dye, the tip of a pipette was carefully pushed against the vitelline membrane so as to insert it into the perivitelline space without piercing the cellular layer. An Eppendorf injector (FemtoJet 5247, Eppendorf) was used with the following settings: compensation pressure: 10 hPa, injection pressure: 220 hPa. Typically, the injection was performed using the ‘clean’ command such that maximal pressure was applied.
After injection, the embryos were cut along the side opposite to the site of injection for subsequent cantilever insertion.
8
RNAi Knockdown of Duox and TLR3 in Larvae
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The BsfDuox and BsfTLR3 sequence fragments were amplified with PCR with premier 5.0 to design of specific primers conjugated with the T7 RNA polymerase promoter. The primer pairs used in dsRNA synthesis are shown in S1 Table .1 μg PCR product was used as the template for dsRNA synthesis utilizing the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA). The dsRNA was isopropanol-precipitated overnight, resuspended in RNase-free H2O, and quantified at 260 nm by using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific Inc., Waltham, MA, USA) before microinjection. The quality and integrity of dsRNA were determined by agarose gel electrophoresis. The injection condition was set to Pi of 300 hpa and Ti of 0.3 s using an Eppendorf micromanipulation system (Microinjector for cell biology, FemtoJet 5247, Hamburg, Germany). Gene silencing experiments of Duox-TLR3 RNAi and egfp RNAi larvae were performed by injecting 1 μl of 2 μg/μl dsDuox-RNA and dsTLR3-RNA solution as well as dsegfp-RNA into the abdomen of each larva.
9
Zebrafish Embryo Xenotransplantation Assay
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Zebrafish embryos were raised at 28°C under standard experimental conditions. Zebrafish embryos at the age of 24 hpf (hours post‐fertilization) were incubated in water containing 0.2 mmol/l 1‐phenyl‐2‐thio‐urea (PTU, Sigma). At 48‐hpf, zebrafish embryos were dechorionated with a pair of sharp‐tip forceps and anesthetized with 0.04 mg/ml of tricaine (MS‐222, Sigma). Anesthetized embryos were subjected for microinjection. Sorted NK cells were labeled in vitro with 2 µM of Carboxyfluorescein succinimidyl ester (CFSE) and mixed with A549‐tdTomato cells (1:1 ratio) and injected at 5 nl (approximately total 500 cells) into the perivitelline space (PVS) of each embryo by an Eppendorf microinjector (FemtoJet 5247, Eppendorf and Manipulator MM33‐Right, Märzhäuser Wetziar). Non‐filamentous borosilicate glass capillaries needles were used for injection and the injected zebrafish embryos were immediately transferred into PTU aquarium water 33°C until the end of experiment. 24 h after injection, zebrafish embryos were monitored by confocal laser scanning microscope LSM 700 system (Zeiss). Appendix Fig S1 C illustrates the workflow for zebrafish injection.
10
Silkworm Embryo Microinjection Protocol
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A diapausing strain, Dazao, which is wildly used as a wild type silkworm, was utilized in this study. The larvae were reared with fresh mulberry leaves at 25°C, 75% RH. Parental embryos (P) were incubated at 15°C and 75% RH to produce nondiapusing eggs (G0), which are suitable for microinjection. The microinjections were performed utilizing TransferMan NK2 micromanipulator and Femto Jet 5247 microinjector (Eppendorf) under an SZX16 microscope (Olympus) as we reported previously19 (link). The final concentration of each plasmid was 250 ng/uL. Embryos were injected within 2 hours after oviposition. The injection opening was sealed with instant glue (Konishi co.) and injected embryos were incubated at 25°C and 90%RH. Larvae hatched from the injected embryos were collected and reared on fresh mulberry leaves.
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