Pv830

Embryos at one-day post-fertilization (dpf) were microinjected individually using a pneumatic picopump (PV830; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) with a pulled microcapillary pipette-using device (PC-10; Narishigen, Tokyo, Japan). Injection of each rHDL alone (16 μg of apoA-I) or co-injection with CML (500 ng) was performed at the same position in the yolk to minimize bias, as described previously [43 (link),59 (link)]. After the injection, the live embryos were observed under a stereomicroscope (Motic SMZ 168; Hong Kong) and photographed (20× magnification) using a Motic cam2300 CCD camera. At 24 h post-injection, each live embryo was compared after removing the chorion to compare the developmental stage at higher magnification (50×).

Embryos at one-day post-fertilization (dpf) were injected individually by microinjection using a pneumatic picopump (PV830; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) with a pulled microcapillary pipette-using device (PC-10; Narishigen, Tokyo, Japan). The injections were performed at the same position on the yolk to minimize bias, as described elsewhere [60 (link)]. CML (500 ng) alone, CIGB-258 (1 ng), Infliximab (43 ng), Etanercept (15 ng), or Tocilizumab (44 ng) were injected into the flasks of embryos (final volume 5 nL). After the injection, the live embryos were observed under a stereomicroscope (Motic SMZ 168; Hong Kong) and photographed (20× magnification) using a Motic cam2300 CCD camera. The survivability of the embryo was assessed using the OECD guidelines 2013 [61 (link)] with coagulation of the embryo, non-detachment of the tail, lack of somite, and lack of heartbeat. At 24 h post-injection, each live embryo was compared after removing the chorion to compare the developmental stage at higher magnification (50×).

Embryos at one day post-fertilization (dpf) were injected individually by a microinjection using a pneumatic picopump (PV830; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) with a pulled microcapillary pipette-using device (PC-10; Narishigen, Tokyo, Japan). The injections were performed at the same position on the yolk to minimize bias, as described previously [10 (link)]. CML (500 ng) alone, CIGB-258 (1 ng), Infliximab (43 ng), and Tocilizumab (44 ng) were injected into flasks of embryos (final volume 5 nL). After injection, the live embryos were observed under a stereomicroscope (Motic SMZ 168; Hong Kong) and photographed (20× magnification) using a Motic cam2300 CCD camera. At 26 h post-injection, each live embryo was compared after removing chorion to compare the developmental stage at higher magnification (50×).

The embryos at one-day post-fertilization (dpf) were injected individually by a microinjection using a pneumatic picopump (PV830; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) with a pulled microcapillary pipette-using device (PC-10; Narishigen, Tokyo, Japan). The injections were performed at the same position on the yolk to minimize bias. Either α-syn or apoA-I in the lipid-free state were injected into flasks of embryos (final 5 μM, 50 nL). After the injection, live embryos were observed under a stereomicroscope (Motic SMZ 168; Hong Kong) and photographed using a Motic cam2300 CCD camera. (Motic, Hong Kong, China)

Freshly laid eggs from 3–4 AM under darkness were dried in air for 10 min in a desiccator at room temperature. These dried eggs were fixed with a double-sided tape on a cover slip. Sharp pointed (< 20 μm diameter) glass capillaries (10 μL quartz, World Precision Instrument, Sarasota, FL, USA) were prepared with a Narishige magnetic glass microelectrode horizontal puller (model PN30, Tritech Research, Los Angeles, CA, USA) for injection. Collected eggs were injected with micro capillaries using a shutter CO₂ based picopump injector (PV830, World Precision Instrument) under a stereomicroscope (SZX-ILLK200, Olympus). Injection was performed with a volume of 5 nL per egg using a mixture containing Cas9 (500 ng/μL) and sgRNA (50 ng/μL) through the micropyle. All injections were accomplished within 30 min after egg collection including drying time. Treated eggs were then incubated at room temperature for 4 h before they were transferred to a growing chamber (25°C) for hatching.

Current measurements and data acquisition from CHO cells were performed in whole cell configuration using EPC10 patch-clamp amplifier controlled by PatchMaster software (HEKA Elektronik, Lambrecht, Germany). Pipettes having resistances 3–4 MΩ were pulled from borosilicate capillaries (Sutter Instruments, Novato, CA, USA). The pipette solution for whole-cell recordings contained (in mM) 140 KCl, 4 NaCl, 1 CaCl₂, 2 Mg-ATP, 0.05 Na-GTP, 5 EGTA, 10 HEPES (pH 7.3), and the bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose (pH 7.4). For application of the agonist pneumatic picopump PV830 (World Precision Instruments, USA) was used and the cells were continuously perfused with bath solution in the recording/perfusion chamber (RC-27, Warner Instruments Inc., Hamden, CT, USA).

Embryos at one day post-fertilization (dpf) were individually injected with a microinjection using a pneumatic picopump (PV830; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) and a pulled microcapillary pipette-using device (PC-10; Narishigen, Tokyo, Japan). Bias was minimized by performing the injections at the same position on the yolk. When necessary for co-injection, immediately before the injection, the oxLDL (15 ng of protein) was mixed with OSO or SO (final 2%) in 100 nL. After the injection, live embryos were observed under a stereomicroscope (Motic SMZ 168; Hong Kong) and were photographed using a Motic cam2300 CCD camera.