P. gingivalis W83 and P. endodontalis (isolated from clinical sample) were cultivated in Tryptic Soy Broth (TSB – Difco Co.; Detroit, Michigan, USA) or TSA (Tryptic Soy Agar - Difco Co.; Detroit, Michigan, USA), both supplemented with hemin (5 µg/mL), menadione (1 µg/mL), and 2% of Yeast Extract (Difco Co.; Detroit, Michigan, USA). Growth and cultivation were performed under anaerobic conditions (10% CO2, 10% H2, and 80% N2) using an anaerobic chamber (MiniMacs Anaerobic Workstation - Don Whitley Scientific; Shipley, West Yorkshire UK) at 37°C.
Minimacs anaerobic workstation
Manufactured by Don Whitley Scientific
Sourced in United Kingdom
The MiniMACS anaerobic workstation is a self-contained, glovebox-style anaerobic chamber designed for sample manipulation and analysis in an oxygen-free environment. The workstation maintains an anaerobic atmosphere through the use of an airlock and continuous gas purge system.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bhi broth, by BD (1 mentions)
Hemin, by Fujifilm (1 mentions)
Menadione, by Fujifilm (1 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (1 mentions)
Gamma counter, by PerkinElmer (1 mentions)
Wallac 1470 wizard, by PerkinElmer (1 mentions)
8 protocols using minimacs anaerobic workstation
1
Cultivation and Anaerobic Growth of Periodontal Pathogens
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P. gingivalis W83 and P. endodontalis (isolated from clinical sample) were cultivated in Tryptic Soy Broth (TSB – Difco Co.; Detroit, Michigan, USA) or TSA (Tryptic Soy Agar - Difco Co.; Detroit, Michigan, USA), both supplemented with hemin (5 µg/mL), menadione (1 µg/mL), and 2% of Yeast Extract (Difco Co.; Detroit, Michigan, USA). Growth and cultivation were performed under anaerobic conditions (10% CO2, 10% H2, and 80% N2) using an anaerobic chamber (MiniMacs Anaerobic Workstation - Don Whitley Scientific; Shipley, West Yorkshire UK) at 37°C.
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P. gingivalis W83 and P. endodontalis (isolated from clinical sample) were cultivated in Tryptic Soy Broth (TSB – Difco Co.; Detroit, Michigan, USA) or TSA (Tryptic Soy Agar - Difco Co.; Detroit, Michigan, USA), both supplemented with hemin (5 µg/mL), menadione (1 µg/mL), and 2% of Yeast Extract (Difco Co.; Detroit, Michigan, USA). Growth and cultivation were performed under anaerobic conditions (10% CO2, 10% H2, and 80% N2) using an anaerobic chamber (MiniMacs Anaerobic Workstation - Don Whitley Scientific; Shipley, West Yorkshire UK) at 37°C.
2
Cultivation and Maintenance of Oral Bacteria
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All strains used in this study are shown in Table 1 . All Porphyromonas and Prevotella strains were grown in brain heart infusion (BHI) broth (Beckton Dickinson Co., Franklin Lakes, NJ, USA) supplemented with hemin (5 µg/mL) (Fujifilm Wako Chemicals Co., Tokyo, Japan) and menadione (1 µg/mL) (Fujifilm Wako Pure Chemicals, Osaka, Japan), or on BHI blood agar plates (BAP) containing hemin and menadione. Porphyromonas gingivalis strain ATCC 33277 was mainly used for this study to screen the herbal products for anti-bacterial activity as well as to further examine the activity in detail. BHI broth and BHI BAP (without hemin and menadione) were used to maintain strains of the other oral bacteria including streptococci and Aggregatibacter actinomycetmcomitans, Fusobacterium nucleatum. All oral bacteria were grown in an anaerobic chamber (miniMACS anaerobic workstation; Don Whitley Scientific Ltd., Shipley, UK) in 80% N2, 10% H2, and 10% CO2 at 37 °C. A laboratory Escherichia coli strain BW25113, which was used for bacterial membrane potential assay, is maintained in LB (Becton Dickinson) broth and on LB agar under aerobic conditions.
3
Anaerobic Cultivation Technique
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Anaerobic cultivation was realized using the miniMACS anaerobic workstation (Don Whitley Scientific, Shipley, UK) with integrated palladium catalyst, temperature and humidity control. Before entering the workstation two nitrogen flushes were performed to eliminate environmental oxygen. Additionally, absence of oxygen was controlled using anaerobic atmosphere indicator strips (Biomérieux, Marcy-l′Étoile, F).
4
Cultivation and VSCs Assays of Oral Bacteria
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S. moorei (DSM 22971) was kept on Bacto™ Tryptic Soy Agar (TSA; Difco, Le Pont de Claix, France) supplemented with sheep blood (7% v/v). F. nucleatum (NCTC 11326) was kept on TSA supplemented with 0.2% Bacto™ Yeast Extract (YE; Difco, Le Pont de Claix, France), 5 μg/mL of hemin (HE; Sigma, Poole, UK), 1 μg/mL of menadione (ME; Sigma, Poole, UK), and 5% (v/v) sheep blood. The cultures were grown under anaerobic conditions (10% CO2, 10% H2, 80% N2) in an anaerobic chamber (MiniMacs Anaerobic Workstation, Don Whitley Scientific, Shipley, UK) at 37°C. For the VSCs assays, S. moorei was cultured in Bacto™ Tryptic Soy Broth (TSB; Difco, Le Pont de Claix, France), while F. nucleatum was cultured in TSB supplemented with yeast extract, hemin, and menadione.
5
Hypoxic Cell Uptake of Radiotracer [67Ga]Ga-US2
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HT-29, MDA-MB-231, RCC4-VHL, and RCC4-VA cells were incubated in 12-well plates (2 × 105 cells/well) at 37°C in an atmosphere containing 5% CO2 for 24 h. After incubation, cells were then incubated at 37°C in an atmosphere containing 5% CO2 and 21% O2 (normoxic conditions) or 1% O2 (hypoxic conditions) for another 24 h. After removing the medium, [67Ga]Ga-US2 (18 kBq, 50−82 GBq/μmol) in the medium (1 mL) was added to each well inside the hypoxic chamber (miniMACS Anaerobic Workstation; Don Whitley Scientific, West Yorkshire, U.K.), and the plates were incubated under normoxic or hypoxic conditions for 2 h. Nonspecific binding was evaluated by the addition of acetazolamide (50 μM). After incubation, each well was washed with 1 mL of PBS (pH 7.4) (Nacalai Tesque), and the cells were lysed with 1 M NaOH (0.5 mL × 2). Radioactivity bound to cells was measured using a γ counter (PerkinElmer). The protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific).
6
Measuring Hypoxia-Induced Radionuclide Uptake
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HT-29, MDA-MB-231, RCC4-VHL, and RCC4-VA cells were incubated in 12-well plates (2 × 105 cells/well) at 37 ºC in an atmosphere containing 5% CO2 for 24 h. After incubation, cells were then incubated at 37 ºC in an atmosphere containing 5% CO2 and 21% O2 (normoxic conditions) or 1% O2 (hypoxic conditions) for another 24 h. After removing the medium, [111In]US1 or [111In]US2 (20 kBq, 80.2-130.7 GBq/μmol for [111In]US1 and 104.0-112.8 GBq/μmol for [111In]US2) in the medium (1 mL) was added to each well inside the hypoxic chamber (miniMACS Anaerobic Workstation; Don Whitley Scientific, West Yorkshire, U.K.), and the plates were incubated under normoxic or hypoxic conditions for 2 h. Nonspecific binding was evaluated by the addition of acetazolamide (50 μM). After incubation, each well was washed with 1 mL of PBS (pH 7.4) (Thermo Fisher Scientific), and the cells were lysed with 1 M NaOH (0.5 mL × 2). Radioactivity bound to cells was measured using a γ counter (Wallac 1470 Wizard; PerkinElmer, Massachusetts, U.S.A.). The protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific).
7
Anaerobic Growth of P. gingivalis Strains
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P. gingivalis ATCC 33277, W83 and HG66 was kindly provided by the Periodontal Research Group, School of Dentistry, University of Birmingham, UK. Bacteria were grown in liquid medium (Fastidious Anaerobe Broth, LAB M, Lancashire, UK) containing 5% sheep blood agar containing, per litre, 10.0 g yeast extract, 5 mg haemin, 0.5 mg vitamin K1, 500 mg L -cysteine were incubated at 37 °C in an anaerobic atmosphere (miniMACS anaerobic workstation; Don Whitley Scientific, UK) of 10% H2, 10% CO2 and 80% N2. Growth of the P. gingivalis strains was monitored spectrophotometrically over a period of 72 h (optical density at 600 nm) and samples of the cultures were Gram stained to confirm their purity.
8
Effect of Prenylated Flavonoids on P. gingivalis
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