For phenotypic evaluation, C. albicans SC5314, high biofilm formers (HBF) and low biofilm formers (LBF) [49 (link),50 (link)], and E. faecalis ER5/1, were standardized to 1 × 106 cells/mL for C. albicans and 1 × 107 cells/mL for E. faecalis in Todd-Hewitt broth (THB; Merck UK) supplemented with 10 mM menadione and 10 mg/mL hemin (Thermo Fisher). These were subsequently mixed 1:1 v/v with Roswell Park Memorial Institute (RPMI-1640 [Sigma–Aldrich, Dorset, UK]) (THB:RPMI), a media which has been shown to support the co-culture of C. albicans and bacterial species [51 (link)]. Mono-cultures of C. albicans and co-cultures of C. albicans with E. faecalis were created in a 24 well microtiter plates (Corning Incorporated, Corning, NY, USA) for 24 h in 5% CO2 at 37 °C. After incubation, biofilms were washed with PBS and stained with 5 μM calcofluor white (Invitrogen, Paisley, UK), a stain which specifically stains the chitin and beta-glucans of fungal cell wall [52 (link)]. Biofilms were incubated in the dark for 20 min and excess stain was washed with sterile water. 2% paraformaldehyde was used for 1 h to fix the stained biofilms which were then imaged using EVOS FL Cell Imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Biofilm biomass of the same biofilms was also quantified using crystal violet stain as previously described [53 ].
Menadione
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Menadione is a synthetic vitamin K analogue used as a dietary supplement and in laboratory research. It serves as a source of vitamin K activity.
Automatically generated - may contain errors
Lab products found in correlation
Hemin, by Thermo Fisher Scientific (4 mentions)
Cellrox green, by Thermo Fisher Scientific (2 mentions)
Menadione, by Merck Group (2 mentions)
Calcofluor white, by Thermo Fisher Scientific (1 mentions)
24 well microtiter plate, by Corning (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Menadione, by MP Biomedicals (1 mentions)
Trypticase soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Hemin, by Merck Group (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Todd hewitt broth, by Merck Group (1 mentions)
Hoechst stain, by Thermo Fisher Scientific (1 mentions)
Cellrox green probe, by Thermo Fisher Scientific (1 mentions)
Evos m7000 imaging system, by Thermo Fisher Scientific (1 mentions)
Pf9366, by Selleck Chemicals (1 mentions)
M200 plate reader, by Tecan (1 mentions)
9 protocols using menadione
1
Fungal-Bacterial Biofilm Phenotypic Evaluation
2
Quantifying ROS in Lung Cells
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Beas-2B or pSAECs were plated with 5000 and 7500 cells per well, respectively, in clear-bottom, black-walled 96-well microplates and cultured 2 days prior to treatment with incinerated thermoplastics. At designated time points, thermoplastic- and menadione-treated cells were stained with 5 μM of CellROX Green (ThermoFisher Scientific) and 1 μM Hoechst 33342 in complete medium for 30 min under standard culture conditions and visualized using the ImageXpress. Nuclei were visualized using a standard DAPI filter set, while CellROX was visualized using a FITC filter set. Treatment with 100 μM menadione (MP Biomedicals, LLC.; Solon, OH) for 60 min in complete growth medium was used as a positive ROS control prior to ROS assessment. After measurement/imaging, cells were fixed using 4% formaldehyde for 15 min at room temperature. A binary mask was generated based on nuclear Hoechst 33342 intensity from the DAPI channel and overlaid onto each nucleus for single cell identification. An intensity-based mask for the FITC channel was applied covering the nucleus and cytoplasm using the MetaXpress. Cell-specific masks were then quantitated for average intensity prior to comparisons.
3
Purification of Mfa1 and FimA Fimbriae from P. gingivalis Mutants
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Mfa1 and FimA fimbriae were purified from P. gingivalis mutant JI-1 (fimA deleted) [36 (link),37 (link)] and SMF-1 (mfa1 deleted) [38 (link),39 (link)] derived from ATCC 33277. JI-1 and SMF-1 cells were cultured under anaerobic conditions at 37 °C on Brucella HK agar medium (KYOKUTO, Tokyo, Japan) prepared by mixing 5% [volume/volume (v/v)] laked rabbit blood, 2.5 μg/mL hemin (Sigma-Aldrich, St. Louis, MO, USA), 5 μg/mL menadione (Sigma-Aldrich), and distilled water. Liquid medium was prepared by mixing trypticase soy broth (Thermo Fisher Scientific), 0.25% yeast extract (Thermo Fisher Scientific), and distilled water, followed by sterilization and the addition of 2.5 μg/mL hemin and 5 μg/mL menadione.
4
Lactobacillus Inhibits Candida Albicans Biofilm
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The ability of the following 7 Lactobacillus strains to inhibit C. albicans SC5314 biofilm formation was assessed: L. casei ATCC 393, L. fermentum ATCC 14931, L. crispatus ATCC 33820, L. iners DSMZ 13335, L. salivarius ATCC 11741, L. jensenii ATCC 25258 and L. rhamnosus ATCC 7469. Overnight cultures of C. albicans and Lactobacillus were grown in YPD and de Man, Rogosa, and Sharpe (MRS; Merck UK) medium, respectively, under appropriate culture conditions. For these experiments, we used an optimized medium of Todd-Hewitt broth (THB; Merck UK) supplemented with 10 μM menadione and 10 μg/ml hemin (Thermo Fisher) and mixed 1:1 with RPMI (referred to here as 1:1 broth), as described previously for coculture experiments (53 (link)). For biofilm formation, overnight cultures were standardized to 1 × 106 CFU/ml for C. albicans and 1 × 107 CFU/ml for Lactobacillus species in 1:1 medium. Eight biofilms of each C. albicans -Lactobacillus pair were incubated in 5% CO2 for 24 h. In addition, C. albicans biofilms were grown for 4 h prior to Lactobacillus being added for 20 h, with biomass quantified using the crystal violet assay.
5
Quantifying Oxidative Stress in Cardiomyocytes
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The oxidative stress generated by optical and electrical stimulation was measured by a fluorogenic CellROX® green probe (Invitrogen; Carlsbad, CA). Four different groups of NRVMs were seeded on 24-well plates. One group was photostimulated for 3 min using 1 Hz light pulse. The second group was electrically stimulated using the same frequency. Finally, a negative (untreated cells) and positive [treated with 200 μM menadione for 1 h (Sigma-Aldrich, St Louis, MO, USA] control were performed with the third and fourth group. Cells, after stimulation and menadione treatment, were incubated in 5 μM CellROX® green and 20 ng ml−1 Hoechst stain (ThermoFisher Scientific, Waltham, MA, USA) for 30 min. Samples were rinsed with PBS, fixed in 4% PFA, and imaged immediately using an EVOS M7000 Imaging System (ThermoFisher Scientific, Waltham, MA, USA). CellROX® intensity values were normalized to the number of Hoechst-positive cells and corrected for background fluorescent intensity of cells and NRVMs without CellROX®.
6
Infection of hGFs by P. gingivalis
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P. gingivalis (ATCC 33,277) was cultured in the brain heart infusion (BHI; Oxiod, USA) medium supplemented with hemin (5 mg/L; Alfa Aesar, USA) and menadione (1 mg/L; Alfa Aesar, USA) in the anaerobic condition (85% N2, 5% H2, and 10% CO2). The bacteria were obtained by centrifugation, washed by PBS twice, and re-suspended in DMEM. The concentration of bacteria was determined by the spectrophotometer at 600 nm with an OD value of 1 equal to 1 × 10^9 CFU/mL. The hGFs were infected with P. gingivalis at the exponential proliferation stage. The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 105 cells/2 mL per well before infection.
7
LAB Supernatant Inhibition of PA14 Biofilms
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PA14 biofilms were grown as described above. Filtered LAB supernatants from 24 h LAB cultures were generated as described in the agar-well diffusion assay, and then serially diluted 2× into PBS pH 7.4. The supernatant from the biofilm cultures was removed and 250 µL of diluted LAB supernatants or buffered MRS control was added to each well and the plates were incubated at 37 °C for 24 h. The supernatant was then removed and the plates were washed 2× with PBS to remove nonadherent cells. 200 µL of LB and 100 µL of XTT solution (0.4 mg/mL XTT, Amresco; 50 µM menadione, Alfa Aesar) were added to each well and plates were incubated at 37 °C for 3 h. Microplates were centrifuged at 3000 × g for 10 min and 200 µL of solution was aliquoted into a fresh microplate. The absorbance at 475 nm was taken to determine viability and this value is reported in the corresponding figures.
8
Thermal Decomposition of Lawsone Complexes
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All solvents were of analytical grade and used without further purification. Dry solvents were used for synthesis and stored under argon. Lawsone, menadione (Acros Organics), RuCl -16). The recorded TGA curves revealed thermal decomposition at T < 100 °C, which did not allow the determination of the water content of the samples by this method.
9
Redox Activity Measurement using WST-8 and Menadione
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To measure redox activity, 17 mg of WST-8 (GenScript, Piscataway, USA) and 13.8 mg of menadione (2-methyl-1,4-naphthoquinone; ACROS Organics, Geel, Belgium) were dissolved in 2.43 ml nanopure water and 10 ml DMSO, respectively, to obtain concentrations of 10 mM and 8 mM. Stock solutions were stored at -20°C. A detection reagent was prepared by mixing WST-8, menadione and water at ratios of 9:1:10. 20 µl of this solution was aliquoted into the wells of a 96 well plate, followed by addition of 100 µl of TSB. The reaction was started from addition of 80 µl of cell suspension using a multichannel pipette. After thorough mixing by pipetting up and down several times, plates were immediately transferred to a TECAN M200 plate reader (TECAN, Austria). Signals were measured at 460 nm at time 0 and after 1, 6, 12 and 24 hours. Prior to each measurement, plates were shaken for 5 sec (linear shaking using an amplitude of 3).
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