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Application of NMDA (100 μM) elicited similar increases in cytoplasmic Ca2+, measured using the cytoplasmic indicator GCaMP2 (Fig. 2A).
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The cytoplasmic extracts (cytosol) were prepared using the Cytoplasmic Extraction Reagent (Pierce) according to the manufacturer's instructions.
Cytoplasmic Ca2+ concentration were determined using the calcium indicator Fura-2AM (Molecular Probes, Life Technologies).
Mitochondrial ROS was detected using the fluorescence indicator MitoSOX (Life Technologies Inc.).
The constraints including 'if' indicators are implemented directly using the GAMS/CPLEX indicator constraint facility.
In addition, measuring cytoplasmic Ca2+ levels by using the fluorescent Ca2+ indicator indo-1 proved that spermatozoa hyperactivation is potentially regulated by Ca2+ influx.
Nuclear and cytoplasmic fractionations were performed using the Nuclear and Cytoplasmic Extraction Kit (Pierce).
Cytoplasmic proteins were extracted using the N-PER cytoplasmic protein extraction kit according to manufacturer's instructions.
To isolate cytoplasmic and nuclear proteins, cytoplasmic and nuclear extracts were prepared using the Nuclear and Cytoplasmic Extraction kit (Beyotime, Nantong, China) according to the manufacturer's instructions.
Proteins from the nuclear and cytoplasmic fractions were isolated by using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific).
Cytoplasmic and nuclear proteins were extracted using the NE-PER NuCytoplasmicytoplasmic Extraction Reagent kit (Pierce Biotechnology) according to the manufacturer's instructions.
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