Du530 spectrophotometer

Axenic culture of C. burnetii was performed using acidified citrate cysteine medium (ACCM-1) (9 (link)) (in the absence of the steroid-binding compound methyl-β-cyclodextrin) at 37°C under microaerobic conditions (5% CO₂ and 5% O₂). All cultures were incubated in T-25 cell culture flasks unless otherwise specified, and growth quantified by measuring culture optical density at 600 nm (OD₆₀₀) using a Beckman DU530 spectrophotometer (Beckman Coulter, Indianapolis, IN). Progesterone (P4), 5-pregnen-3β-ol-20-one (pregnenolone [P5]), or 17β-estradiol (E2) (Sigma-Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO) and diluted in the culture medium to specified concentrations. The concentration of DMSO was normalized across all conditions to control for any effects of the solvent on bacterial replication. Analyses of the effect of hormones on pathogen replication were conducted with the concentration of DMSO below inhibitory concentrations. Bacterial viability was measured using a CFU assay. Ten-microliter samples were spotted in triplicates on ACCM-2 plus tryptophan (0.5 mM) as described previously (40 (link)). Samples were stored at −80°C in ACCM-2 plus 10% DMSO until analysis.

To determine the effects of estradiol and progesterone on endometrial epithelial monolayer permeability, eEC were cultured in 1-μm Millicell hanging cell culture inserts (EMD Millipore) coated with Matrigel (BD Biosciences) with donor-paired eSF in the basolateral chamber as described [14 (link)]. In all samples tested, eEC were confirmed to form an intact barrier prior to exposure to hormones as assessed by transepithelial resistance and leakage of phenol red (32 μg/ml) [14 (link)]. To monitor the effects of estradiol and progesterone on barrier function, the basolateral chambers of the cultures were then exposed to vehicle, 10 nM estradiol, or 10 nM estradiol + 1 μM progesterone for 6 days, with basolateral media replenished half way through the assay, similar to methods described [15 , 57 (link)]. At the end of 6 d, 100 μl of apical and basolateral media were collected and assessed for absorbance at 559 nm using a Beckman Coulter DU 530 spectrophotometer (Beckman Coulter). Absorbance values of culture medium lacking phenol red served as background control, while absorbance values of culture media containing 32 μg/ml of phenol red served as the input control. For each donor, % leakage was calculated by the following formula: (OD₅₅₉(basolateral)–OD₅₅₉(background)) / ((OD₅₅₉(input)—OD₅₅₉(background)) x 100.

Non-enzymatic browning was measured according to the method of [24 (link)] with slight modification. One mL of sauce was stirred using a magnetic stir bar and plate with 10 mL of ethanol (500 mL L⁻¹v/v Fisher Scientific, Waltham, MA, USA) for one hour. The mixture was then filtered through a 0.45 μm syringe filter (MDI Membrane, Harrisburg, PA, USA) and subjected to an absorbance measurement at 420 nm with a DU 530 spectrophotometer (Beckman Coulter, Brea, CA, USA).

Total RNA was prepared from 100 adult male or female An. anthropophagus mosquitoes using trizol (Invitrogen) according to the manufacturer’s protocol. The isolation methods of total RNA and small RNA were unbiased in each sample. All samples were ground in liquid nitrogen and the quality of RNA was detected by using denaturalization agar gels and Du-530 Spectrophotometer (Beckman, Gemany).
The RNA smaller than 200 bp were enriched with the mirVana miRNA isolation kit (Ambion, USA). The small RNA samples were sent to Genergy Bio. (Shanghai, China) for small RNA cloning. The population of miRNAs with a length of 15–30 bp was passively eluted from polyacrylamide gels. The RNA was then precipitated with ethanol and dissolved in water. The small RNAs collected had a poly(A)- tail added to their 3'–OH by poly-(A) polymerase. The 5′-phosphate of the small RNAs were ligated to an RNA adapter. First-strand cDNA synthesis was then performed using an oligo(dT)-linker primer and MMLV-RNase H reverse transcriptase (Promega, USA). The resulting cDNAs were PCR amplified to ~ 25 μg/μl.

Total collagen content in the colon was detected with Sirius red collagen detection kit (Chondrex, Redmond, WA, USA). Colonic tissue was homogenized in T-PER buffer (Thermal Science, Amarillo, TX, USA) using a TissueLyser (Qiagen, Germantown, MD, USA), incubated on ice for 15 min, and centrifuged for 5 min at 10,600 × g at 4°C (Heraeus™ Primo™/Primo R centrifuge; Thermo Scientific, Waltham, MA, USA). Each protein sample was diluted in 0.5 M acetic acid to a final concentration of 100 µg/ml. Optical density was read at 530 nm against the reagent blank using a DU-530 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA, USA). Results were calculated based on collagen per 100 µg/ml protein. Cultured SEMFs were characterized by immunohistochemistry. Mouse monoclonal antibodies against α-SMA, vimentin and desmin were used (Sigma). The cells were grown on glass coverslips and fixed using acetone, prior to immunoperoxidase staining with the Vectastain ABC peroxidase kit (Vecta Laboratories, Burlingame, CA, USA). Following incubation with the primary antibody, biotinylated goat anti-mouse immunoglobulin(Ig)-G (Sigma) was applied and subsequently avidin-biotinylated horseradish peroxidase complex. Peroxidase activity was developed with diaminobenzidine, followed by nuclear staining using hematoxylin (Sigma).