Microfuge 20r centrifuge

To perform protein isolation, a culture medium was removed from culture flasks; the cells (approximately 3 million cells per one culture flask) were washed twice using ice-cold PBS and lysed with 500 μL of a radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplied with a Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. The obtained cell lysate was centrifuged using a Microfuge 20R centrifuge (Beckman Coulter, Brea, CA, USA) for 15 min at 14,000× g and 4 °C; the supernatant was transferred into a clean 1.5 mL Eppendorf tube. The quantity of the isolated protein was evaluated via a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) using a Multiskan Sky microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

BALF was collected according to the routine bronchoscope operating procedure: 20 mL of normal sterile saline, warmed to body temperature (37°C), was injected for bronchoalveolar lavage. BALF volume of 5–10 mL was recovered and sent for microbial inspection in a disposable sterile silicon plastic bottle. Microbial inspection was performed using an ELx808IU microplate reader (BIoTek, Winooski, VT, United States). The recovered BALF was transferred to a 10-mL centrifuge tube using a disposable sterile pipette. After centrifugation at 1,000 rpm for 10 min using a microfuge 20R centrifuge (Beckman Coulter, Brea, CA, United States), 600–800 μL of supernatant was collected and stored at –40°C for further tests. Venous blood (3 mL) was collected and centrifuged at 3,500 rpm for 10 min; serum was collected and stored at 4°C.
The GM test was performed to detect GM levels in serum and BALF samples using the one-step enzyme immunoassay sandwich method (Aspergillus antigen detection kit). The BDG test was mainly performed to detect BDG in serum and BALF samples using the dynamic turbidimetric method (fungal dextran detection kit). All tests were conducted following the manufacturer’s instructions.
Serum GM > 0.5, BALF GM > 0.7, serum BDG > 100 pg/mL, and BALF BDG > 200 pg/mL were defined as positive values for fungal infection (9 (link), 20 (link)).

For the determination of total protein content, extraction was performed as described by [13 ] with some modifications. One gram of plant samples (inoculated and healthy leaves) were crushed separately in a pestle and homogenized in 5 ml of the Tris-Maleate buffer (Tris-cHcl 1 HCl 10 mM, Triton X-100 1%, pH 7.5) at 4°C. The homogenate was centrifuged at 10,000 ×g for 25 min at 4°C using the Beckman-Coulter Microfuge 20R centrifuge. The supernatant was collected and the precipitate re-suspended in 3 ml buffer followed by further centrifugation. The second supernatant was collected, mixed with the first in 1.5 ml Eppendorf tubes and stored at -20°C. Quantification of the concentration of total proteins was done using the standard Bradford assay. The absorbance was measured at 595 nm using a UV-Vis 1605 Shimadzu spectrophotometer. For each extract, three repetitions were carried out. Bovine Serum Albumin was used as the standard. The concentration of the protein present was expressed in mg/g of fresh matter.