Geldoc go gel imaging system

Inactivated SARS-CoV-2 (0.1 mL, 1.3 × 10⁷ PFU/mL) was diluted to 1 mL in water. The filter efficiency test was repeated (n = 3) following the procedure as described above. However, the syringe filters were 13 mm instead of 30 mm. In a similar fashion, these were used to collect aerosolised material (in this case inactivated SARS-CoV-2) and a pre-prepared commercial extraction fluid (0.4 mL) was used as provided with a FlowFlex SARS CoV-2 Antigen Rapid Test (lateral flow assay kit). The lateral flow assays used were from the same LOT and factory number, to minimise variability. 0.1 mL of the extract was recovered through the syringe filters and dripped onto the SARS-CoV-2 antigen rapid test strip. It was then allowed to rest for 30 min as per the manufacturer’s instructions (Fig. 7). The test strips were analysed using a GelDoc Go Gel Imaging System (BioRad, CA, USA) and the intensity of the control and test strip lines were recorded and analysed using Image Lab software (version 3.0.0.07, BioRad, CA, USA). A calibration curve of 13 different concentrations of SARS-CoV-2 between 1.3 × 10² and 6.5 × 10⁵ PFU/mL was produced to allow for conversion of test line intensity to concentration (Supplementary Fig. S6).

Following a 24-h incubation of the cells with various nanomaterial groups, RIPA lysis buffer (2 mL) was added to lyse the cells. The lysis buffer contained 20 μl of 1% protease inhibitor and 20 μl of 1% 0.5 M EDTA per mL of RIPA. After 1 h of lysis, the lysate was centrifuged at 8050 x g for 10 min, and the supernatant was collected for further analysis. Cell proteins were separated by SDS-PAGE gradient gel and transferred onto a PVDF membrane. Protein loading buffer was added to the samples, and they were thoroughly mixed and denatured at 98 °C for 10 min. GLUT-1 protein was not denatured since Abcam’s instructions mention that GLUT-1 protein may aggregate irreversibly after boiling. Avoiding boiling helps prevent GLUT-1 proteins from entering the gel or forming polymers that are often mistaken for background. Our experimental design takes this factor into consideration to ensure the accuracy and reliability of the experimental results. The PVDF membranes were incubated overnight at 4 °C with primary antibodies against GLUT-1 (dilution: 1:100000; Abcam, Cambridge, UK), RAD51 (Abcam, ab133534, 1: 1000 dilution), and β-Actin (dilution: 1:1000; Abcam, Cambridge, UK). Subsequently, the membranes were incubated with the corresponding secondary antibodies at 37 °C for 1 h. Protein bands were visualized using the GelDoc Go Gel Imaging System (Bio-RAD).

Headloop PCR was done as previously described (Rand et al., 2005 (link); Selvaraj et al., 2019 (link)). Assessment of PCR products on agarose gel is sufficient to determine whether the target loci were effective mutants in F0 embryos. The headloop PCR primer pairs and the headloop tags are provided in Additional file 4: File S3. For headloop PCR, each well contained: 2 μl 5× Phusion HF buffer, 0.2 μl dNTPs (10 mM), 0.05 μl forward primer (10 μM), 0.05 μl reverse primer (10 μM), 0.1 μl Phusion Hot Start II polymerase, 1 μl genomic DNA, 6.6 μl dH₂O; for a total of 10.0 μl. PCR program was: 98°C for 30 s; then 30 cycles of: 98°C for 10 s, 72°C for 20 s; then 72°C for 7 min. Amplification was assessed by agarose gel electrophoresis (Figure 7Da). To calculate the headloop PCR score, GelDoc Go gel imaging system (Bio-Rad) was used to imaging the gel. Then, Quantity One software (Bio-Rad) was used to quantify band intensity.