Oocytes still enclosed in the cumulus cells (CEOs) were injected with 12.5 ng/μl Il7-RL, Thumpd1-RL, or Tpx2-RL and 12.5 ng/µl of polyadenylated FL using a FemtoJet Express programmable microinjector with an Automated Upright Microscope System (Leica, DM4000B). Injected CEOs were pre-incubated for 3 h in culture medium supplemented with 2 μM milrinone, and then cultured in milrinone-free medium supplemented with 100 nM amphiregulin. After 16 h, the CEOs were denuded, collected in lysis buffer and frozen. Luciferase activities in the oocyte extracts were measured using a dual-luciferase reporter assay kit (Promega), and luminescence was detected with a SpectraMax L luminometer (Molecular Devices). Data are reported as ratios of RL and FL.
Femtojet express
Manufactured by Leica
The FemtoJet Express is a microinjection system designed for precise and consistent cell manipulation. It features a compact and streamlined design, providing a reliable solution for various cell-based applications.
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Lab products found in correlation
2 protocols using femtojet express
1
Microinjection of Oocytes with Regulators
2
Live-cell Imaging of Translational Reporters
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Collected GV-arrested oocytes were injected with 5–10 pl of 12.5 ng/μl solution of the Ypet reporters along with a mCherry reporter or FRET reporter using a FemtoJet Express programmable microinjector with an Automated Leica microinjection Microscope System (LEICA DMI 4000 B). After overnight pre-incubation in α-MEM with 1 μM cilostamide, oocytes were released from cilostamide and matured in vitro under time-lapse microscopy. Live cell imaging was performed under a Nikon Eclipse T2000-E equipped with a mobile stage and an environmental chamber at 37°C and 5% CO2. Filter set: dichroic mirror YFP/ CFP/ mCherry 69008BS; Ypet channel (Ex: s500/20× 49057 Em: D535/30m 4728811); mCherry channel (Ex: s580/25× 49829 Em: D632/60m); Cerulean channel (Ex: 430/25× 49829 Em: 480/40m 49287), for FRET channel (Ex: 430/25× 49829 Em: D535/30m 47281). Images were processed and fluorescence was quantified using MetaMorph (Molecular Devices). For reporter assay, YFP and mCherry channels were corrected by background. Then, the ratio of Ypet fluorescence and the maximum level of mCherry signal was plotted as an accumulation of translation activity. For the FRET assay, YFP, CFP and FRET channels were corrected by background. FRET channel was corrected for the YFP bleed-trough, and FRET was calculated as FRET channel/CFP channel.
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