Approximately 0.1 ml of filtered and non-filtered aliquots from each samples were inoculated in wells and incubated for 7 days at 40 °C under aerobic conditions.
For γ-irradiation, 140 ml of filtered HS or filtered FBS was separated into seven fractions of 20 ml each.
The filter membrane holes were cleaned with 100 mL of filtered pure water and then water samples were filtered.
To analyze the pigment ratio for each preservation period, 10 mL of floc samples were taken and poured into 100 mL of filtered sea water.
Sodium hydroxide (0.5 M) was slowly added to 10 ml of filtered wetland water while monitoring pH until a macroscopic precipitate was observed.
Each 250 mL of filtered sewage sample were spiked with 100 μL of a surrogate standard solution prepared in methanol (bupropion-d9 / 10,11-dihydrocarbamazepine 10,11-dihydrocarbamazepine 2.5 mg/Lf methandl before lowering the pH to addition with 100 μL of phosphoric acid (85%).
Spawned sperms (200 μl) were added to 2 ml of the experimental medium each containing AgNPs at different concentrations (1, 2, 3, 4, and 5 nM) and kept for 30 min; 500 μl of this treated sperms from each concentration was pipetted into 2 ml of filtered seawater containing untreated eggs (50 eggs), mixed, and observed at intervals of 10 min under microscope.
Control animals were injected with 0.5 mL of filtered seawater.
The membrane was blocked in 10 ml of filtered blocking solution [PBS, 0.05% Tween 20, 5% Non fat dried milk, NFDM] for 1 2 hours with gentle shaking at 4°C.
As internal reference, 1 µL of a freshly prepared dilution of the beads solution (1 drop in 5 mL of filtered distilled water) was added to each culture sample.
One ml of filtered culture medium containing the shRNA-lentivirus was added to 1×105 CNE2 cells with polybrene (Sigma) at a final concentration of 8 µg/µl and after 24h was replaced with medium containing 2 µg/ml puromycin.
