Pes syringe filter

Plasma samples from human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients were obtained from BioIVT (Westbury, NY, USA). Whole blood samples from different healthy donors were purchased Research Blood Components (Watertown, MA, USA). To obtain plasma from whole blood samples, whole blood was centrifuged at 2500× g for 15 min. The supernatant was collected and centrifuged again to obtain the plasma. Both collected and purchased plasma samples were diluted with sterile PBS and filtered with a 0.2 µm PES syringe filter (VWR) before use. The plasma and whole blood samples were not specifically collected for our research and we did not have access to the identifying information of the subjects. Based on U.S. Department of Health & Human Services regulations, our research is non-human subjects research.

Seeds of each pea line were planted, one per square pot, and half of the seedlings were inoculated. All plants were harvested 24 days after planting, i.e., inoculated plants were harvested 21 DAI. On harvest day, it was ensured that no contamination had occurred between non-inoculated and inoculated plants. Shoots and roots were separated, weighed, chopped, and individually placed in 50 mL conical centrifuge tubes. Samples from the same pea line were pooled and covered with ~20 mL of ethyl acetate; they were left to macerate at 4 °C in the dark for 72 h at which time the ethyl acetate was removed and placed in 250 mL round-bottom flasks. A volume of sterile water equal to the weight of the tissue (v:w) was added to the maceration fluid, which was then evaporated under a stream of argon. The aqueous extract left behind was then filter-sterilized using a PES syringe filter with a pore size of 0.2 μm (VWR International, Radnor, PA, USA). The different extracts were either used immediately or stored in 50 mL conical tubes at −20 °C for later use. Before use, the extracts were diluted 20 times in sterile water and on the treatment days, 3 mL of the diluted solution were applied at the soil surface.