Spectramax i3 platform

HCS LIVE/DEAD^® Green Kit (Catalog no. H10290, Invitrogen, Waltham, MA, USA) with a two-color nuclear fluorescence staining assay is used to evaluate the cell viability after PM_2.5 exposure [22 (link)]. The kit includes Image-iT DEAD Green™ viability stain for discrimination of dead cells and HCS NuclearMask™ Deep Red stain for total cell demarcation. The Image-iT^® DEAD Green™ viability stain in the kit is impermeable to healthy cells, whereas it can enter once the membrane integrity of the cells is compromised. High-content fluorescence imaging was performed using the SpectraMax i3 platform with MiniMax Imaging Cytometer (Molecular Devices, Silicon Valley, CA, USA).

Genomic DNA was extracted from stably transfected cells using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) and the Tissue and Cell Genomic DNA Purification Kit (GeneMark). Global DNA methylation levels were determined using MethylFlash^™ Global DNA Methylation (5‐methylcytosine, 5‐mC) ELISA Easy Kit (EPIGENTEK, Farmingdale, NY) in accordance with the manufacturer's instruction. Briefly, the methylated DNA was detected by using capture and detection antibodies to (5‐mC) and then quantified calorimetrically using SpectraMax^® i3 Platform (Molecular Devices, Sunnyvale, CA, USA) to read the absorbance at 450 nm. The amount of methylated DNA was proportional to the measured OD intensity. A standard curve was used to quantify the absolute amount of methylated DNA (the percentage of 5‐mC) in the total genomic DNA. Each sample was assessed in duplicate.

Overnight cultures were diluted to an OD₆₀₀ of 0.05 in LB medium, and 100-μl aliquots were dispensed in a flat-bottom polystyrene 96-well microtiter plate (catalog no. 655180; Greiner Bio-One). Plates containing six technical replicates of each strain tested were incubated statically at 28°C for 48 h. Nonadherent cells were gently washed out of the plate with distilled water. Adherent cells were then stained with 150 μl crystal violet (0.1%) for 15 min, thoroughly rinsed with distilled water, and submerged in 200 μl ethanol-acetone (4:1) for 15 min, after which adherent cells were suspended by pipetting. The quantity of adherent biomass was determined by measuring the absorbance of fixed crystal violet (A₅₉₅) using a SpectraMAX i3 platform operated by SoftMAX Pro 3 (Molecular Devices).

LPO levels were measured using a commercial kit (Cayman Chemical Co., USA) according to the manufacturer’s instructions. Briefly, overnight cultures of C. jejuni strains on MH agar were suspended in MH broth to an OD₆₀₀ of 0.1. The bacterial suspension (3 mL) in a 19-mL glass culture tube was incubated at 42°C with shaking (200 rpm) under aerobic and microaerobic conditions for 6 h. After exposure to each condition, bacterial cultures were centrifuged at 10,000 × g for 5 min. LPOs were extracted with chloroform and methanol and mixed with the Chromogen reagent. After incubation at room temperature for 5 min, the OD at 500 nm was measured with a SpectraMax i3 platform (Molecular Devices, USA). A standard curve was generated with 13-hydroperoxy-octadecadienoic acid. The results were normalized with the protein concentration of each sample that was measured with the Bradford assay.

Broth microdilution assay was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations (http://www.eucast.org, accessed on 18 January 2021). Before the experiments, the bacteria were grown overnight on Mueller Hinton (Sigma-Aldrich, Steinheim, Germany) agar at 36 °C. For the experiment, bacterial suspensions (prepared in Ringer’s solution and equal to 0.5 of McFarland standard (10⁸ CFU mL⁻¹)) were diluted to obtain ~1 × 10⁶ CFU mL⁻¹. The assay was performed in 96-well sterile microplates (Eppendorf, Hamburg, Germany). The extracts and fractions were diluted in 2% v/v DMSO and tested in triplicates at concentrations of 500, 250, 100 and 10 μg mL⁻¹. The fractions were prepared using a two-fold serial microdilution method. The concentration of the fractions used in the experiment ranged from 1.95 µg mL⁻¹ to 1000 µg mL⁻¹. The 96-well microplates were incubated overnight (Gram− for 16 h, Gram+ for 24 h) at 36 °C. The optical density (OP) of each well was measured at 620 nm using SpectraMax^® i3 Platform (Molecular Devices, San Jose, CA, USA). The percentage of growth inhibition was calculated in comparison to the control (bacterial culture without the extract/fraction).

ROS levels were measured as described previously, with slight modifications (55 (link)). The total ROS accumulation level was measured using the fluorescent dye CM-H₂DCFDA (chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate) (Thermo Fisher Scientific, OH, USA). C. jejuni was prepared as a culture grown overnight on MH agar and resuspended in MH broth to an OD₆₀₀ of 0.1. The bacterial suspension was transferred to a disposable culture tube (Kimble, NJ, USA) and incubated at 4°C. Samples were taken before and after exposure to cold stress for 4 days. After treatment with 10 μM CM-H₂DCFDA dye for 30 min at room temperature, fluorescence was measured using a SpectraMax i3 platform (Molecular Devices, CA, USA) at 495 nm excitation and 527 nm emission wavelengths. The fluorescence levels were normalized to the protein amounts determined using the Bradford assay (Bio-Rad, CA, USA).

Cells were seeded in a 48-well cell culture plate 18–24 hr before transfection so that cell density was approximately 70% at time of transfection. LipoD293 DNA In Vitro Transfection Reagent (SL100668 SignaGen Laboratories) was used to transiently transfect cells with a reporter plasmid, expression vectors, and a plasmid expressing Renilla luciferase. LipoD293/DNA complex-containing medium was replaced with recommended culturing medium supplemented with 1% FBS 16–18 hr post-transfection. Drugs were added to cells 48 hr post transfection. Drug-containing medium was removed 16 hr later and cells were washed with 1x PBS. Luciferase activity was assayed using Dual-Glo Luciferase Assay System (E2940 Promega) and in accordance with the Dual-Glo protocol except that a 1:1 mixture of 1xPBS:Dual-Glo Reagent was added to the cells. Luminescence was measured on the SpectraMax i3 Platform (Molecular Devices). See Key Resources for list of plasmids.