Fluorescent microplate reader

Mitochondria were isolated from the fresh ovarian tissue. Tissue was homogenized in an isolation buffer, the homogenate was then subjected to differential centrifugation as previously described^{21 (link)}. Mitochondrial protein at a concentration of 0.4 mg/ml was used for mitochondrial function analysis. Isolated mitochondria were incubated with 2 μM DCFH-DA dye (Sigma-Aldrich, USA) for 20 min at room temperature to determine mitochondrial oxidative stress levels. The fluorescence intensity of DCF was measured using a fluorescent microplate reader (BioTek, USA). We also determined the changes in mitochondrial membrane potential using JC-1 dye. Isolated mitochondria were incubated with 300 nM JC-1 dye (ThermoFisher, USA) for 30 min at 37 °C. Red (J-aggregates: healthy mitochondria) and green (J-monomers) fluorescence intensity were measured using a fluorescent microplate reader (BioTek, USA). Red/Green fluorescent intensity ratio was used to represent the changes in mitochondrial membrane potential. In addition, mitochondrial swelling was measured by change of mitochondrial absorbance over time, which was calculated by mitochondrial absorbance at min x/mitochondrial absorbance at min 0. The data were plotted throughout the 30 min of measurement.

The detection of ASFV by probe-based qPCR has been described previously [29 (link)–31 (link)]. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay. LbCas12a (New England Biolabs) was used for this assay. As described in the DETECTR method, our CRISPR-Cas12a assay also uses recombinase polymerase amplification (RPA). For RPA reactions, the TwistAmp Basic kit (TwistDx) was used according to the manufacturer’s instructions (https://www.twistdx.co.uk/en). Briefly, 50 μl reactions containing 2.5 μl ASFV DNA, 0.48 μM forward and reverse primers, 1× rehydration buffer, 14 mM magnesium acetate and RPA mix were incubated at 39 °C for 20 min. CRISPR-Cas12a detection was performed as described previously with minor modifications. The reaction volume was a total of 50 μl, with 20 nM crRNA, 115 nM single-stranded DNA fluorophore quencher - labeled reporter (Table S1), 30 nM LbCas12a, 1 μl RPA amplification products, 2 μl RNase inhibitor (New England Biolabs), and 1 × LbCas12a Buffer (New England Biolabs). The reactions were incubated in a temperature-controlled water bath for 15 min at 37 °C. Fluorescence emission was excited at 485 nm and detected at 535 nm using a fluorescent microplate reader (BioTek), and reactions without target DNA were used to establish the background.

Mitochondrial ROS production was assessed using a cell-permeable fluorogenic probe 2′,7′-dichlorofluorescein diacetate (DCFDA). DCFDA can diffuse through the mitochondrial membrane and is deacetylated into membrane-impermeable non-fluorescent dichlorofluorescein (DCFH) by cellular esterases. In the presence of ROS in the mitochondria, DCFH is rapidly oxidized to highly fluorescent dichlorofluorescein (DCF). The fluorescent intensity is proportional to the amount of ROS produced by mitochondria [21 (link)]. After incubation with 2 µM DCFDA for 60 min at 25 °C, fluorescence was measured using a fluorescent microplate reader (BIOTEK^® Instruments Inc., Winooski, VT, USA) with excitation at 485 nm and emission at 530 nm.