Gen5 microplate reader

DNA was extracted from lines in the A-set and V-set for GBS using BioSprint 96 DNA Plant Kits (Qiagen, Hombrechtikon, Switzerland) from 2 to 3 leaves of 2-weeks-old seedlings. For the SSR marker test, DNA from a bulk of six leaves of 5 days old plants was extracted using DNAzol Reagent (Molecular Research Center, Inc. Technical Bulletin 6). The tissue was ground using liquid nitrogen then 300 μl DNAzol Reagent was added to this powder and left for 5 min at the room temperature. A volume of 300 μl chloroform was added to the previous mix and left at the room temperature for 5 min before it was centrifuged using Fisher Scientific accuSpin Micor 17 centrifuge (Loughborough, United Kingdom) at 12000 × g for 10 min. The washing process was done in three steps by adding three different washing solutions as follows: (1) absolute alcohol, (2) DNAzol + 75% Ethanol and (3) 75% alcohol only. All genotypes were centrifuged using the Fisher Scientific accuSpin Micor 17 centrifuge for 4 min at 5000 × g after each washing step. The extracted DNA was then re-suspended in 150 μl of TE buffer. DNA concentration was measured using spectrophotometry (Gen5^TM microplate reader and image software with Take3^TM micro-volume plates [BioTek, Winooski, VT, United States]).

To assess the effects of different reagents, we seeded NPCs (density: 3 × 10³ cells per well) in a 96-well plate and viability was examined using a Cell Counting Kit-8 (CCK-8; Dojindo Co., Kumamoto, Japan) following the manufacturer's protocols. The NPCs were treated with different concentrations of aspirin and LPS for different time periods. Optical density (OD) was measured at a wavelength of 450 nm using a Gen5 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The glycolysis rate was measured using Glycolysis Assay Kit (Abcam, Cambridge, UK, ab197244). The appropriate number of cells was carefully planted in a 96-well plate. Post-treatment, the supernatant was removed, and the cells were thoroughly washed twice with 100 μL respiratory buffer. Subsequently, 150 μL respiratory buffer was delicately added to each well, followed by the addition of 10 μL glycolysis detection reagent. The glycolysis rate of the cell was detected using a BioTek Gen5 Microplate Reader (Winooski, VT, USA).

A suspension of overnight culture of each strain of Candida spp. (Candida albicans ATCC90028, Candida albicans ATCC10231, Candida albicans clinical strain 6, Candida albicans clinical strain 10, Candida glabrata clinical strain 14, Candida tropicalis clinical strain 21) grown on Sabouraud chloramphenicol agar plates (SAB Dex Chlor) was prepared in sterile saline (NaCl 0.85% w/v). Suspensions were adjusted to 1.0 × 10⁶ CFU/mL (0.5 McFarland) and then diluted to obtain a concentration of 1.0 × 10⁴ CFU/mL in RPMI 1640.
The activity of the extract against Candida strains was determined by microplate assay following the Clinical and Laboratory Standards Institute document M27-A3 [16 ]. A mixture of 100 µL C. albicans suspensions in RPMI broth (final concentration 0.5 × 10³ CFU/mL) and 100 µL of FSM were added to each well of a sterile 96-well microplate (Thermo Scientific™ Sterilin™, Waltham, MA, USA). Plates were incubated at 35 °C aerobically for 24–48 h, and minimum inhibitory concentration values (MICs) were recorded as OD at 490 nm using a microplate reader (Gen5 Microplate Reader, BioTek Instruments, Winooski, VT, USA).