The levels of intracellular ROS in porcine COCs were measured using the DCF-DA method (Molecular Probes, Eugene, OR, USA), as previously described [37 (link)]. After washing thrice with PVA-PBS medium, mature COCs were incubated in IVM II medium containing 5 μM DCF-DA for 20 min at 38.5 °C and 5% CO2. Following incubation, the COCs were washed thrice with 0.1% PVA-PBS, and their fluorescence was recorded using an Olympus DP 70 camera (Olympus, Tokyo, Japan). Mito-SOX (red)-stained cytoplasm was examined using the iRiS™ Digital Cell Image System (Logos Biosystems, Inc., Anyang, Korea). Porcine COCs were washed thrice in PBS-PVA and cultured in 300 μL of IVM II medium mixed with Mito-SOX (500:1, Invitrogen) at 37 °C for 30 min. The COCs were then fixed with 2.5% glutaraldehyde/PBS (v/v) for 1 h and permeabilized with 0.1% Triton X-100 (v/v) for 30 min, followed by staining with Hoechst 33342 (2 mg/mL). The COCs were covered with a cover glass after mounting on glass slides using a mounting solution.
Mito sox
Manufactured by Logos Biosystems
Mito-SOX is a fluorogenic dye used to detect superoxide in the mitochondria of live cells. It is a positively-charged derivative of dihydroethidium that selectively targets the mitochondria.
Automatically generated - may contain errors
2 protocols using mito sox
1
Measuring Intracellular ROS in Porcine COCs
2
Mito-Ca2+ Levels in Oocyte Maturation
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To evaluate mito-Ca2+ levels in maturated and fertilized oocytes, we performed fluorescence staining using Rhod-2 AM (ab142780; Abcam, Cambridge, MA, USA) as a specific
mitochondria calcium detection dye. To identify Rhod-2 fluorescence expression in mature oocytes (44 h after IVM) and fertilized oocytes (3 h or 6 h after IVF), oocytes were washed
with 0.1% PBS-polyvinyl alcohol (PVA) and incubated in the dark at 38.5ºC and 5% CO2 for 15 min in IVF medium supplemented with 100 μM Rhod-2 dye. After Rhod2 treatment,
oocytes were washed three times in 0.1% PVA-PBS (w/v) and Rhod-2 fluorescence was measured using an iRiSTM digital cell imaging system (Logos Biosystems, Anyang, Korea).
Each sample was washed three times in 0.1% PVA-PBS. Washed presumptive zygotes were cultured in 50 μl of IVC medium mixed with 5 μM Mito-SOX (red fluorescence; Life Technologies,
Carlsbad, CA, USA) under mineral oil at 37°C for 30 min. To confirm the mitochondria localization, embryos cultured in 50 μl of IVF medium were mixed with 4 μg/ml Mito-Tracker
(Cell Signaling Technology, Danvers, MA, USA) at 38.5ºC for 20 min. Each sample was washed three times in 0.1% PVA-PBS (Supplementary Fig. 5 : online only). Mito-Tracker (green) and Mito-SOX (red)-positive cytoplasm in cells were detected using an iRiSTM digital cell imaging
system (Logos Biosystems).
mitochondria calcium detection dye. To identify Rhod-2 fluorescence expression in mature oocytes (44 h after IVM) and fertilized oocytes (3 h or 6 h after IVF), oocytes were washed
with 0.1% PBS-polyvinyl alcohol (PVA) and incubated in the dark at 38.5ºC and 5% CO2 for 15 min in IVF medium supplemented with 100 μM Rhod-2 dye. After Rhod2 treatment,
oocytes were washed three times in 0.1% PVA-PBS (w/v) and Rhod-2 fluorescence was measured using an iRiSTM digital cell imaging system (Logos Biosystems, Anyang, Korea).
Each sample was washed three times in 0.1% PVA-PBS. Washed presumptive zygotes were cultured in 50 μl of IVC medium mixed with 5 μM Mito-SOX (red fluorescence; Life Technologies,
Carlsbad, CA, USA) under mineral oil at 37°C for 30 min. To confirm the mitochondria localization, embryos cultured in 50 μl of IVF medium were mixed with 4 μg/ml Mito-Tracker
(Cell Signaling Technology, Danvers, MA, USA) at 38.5ºC for 20 min. Each sample was washed three times in 0.1% PVA-PBS (
system (Logos Biosystems).
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