P. gingivalis ATCC33277 was maintained on blood agar plate containing 40 mg/mL Trypto‐Soya Ager (Nissui Cat# 05516), 5 mg/mL Brain Heart Infusion (Becton Dickinson Cat# 211059), 1 mg/mL L‐Cysteine Hydrochloride (Wako Cat#039‐05274), 1 μg/mL Menadione (Sigma‐AldrichCat# M9429‐25G), 5 μg/mL Hemin (Sigma‐Aldrich Cat# 51280‐1G) and 5% defibrinated sheep blood (ThermoFisher Cat# R54020). When P. gingivalis was ready to use, it was grown in liquid medium containing 37 mg/mL Brain Heart Infusion, 2.5 mg/mL Yeast Extract (Becton Dickinson Cat# 212750), 1 mg/mL Cysteine, 1 μg/mL Menadione and 5 μg/mL Hemin. We used an anaerobic chamber with 10% CO2, 10% H2, and 80% N2 maintaining at 37°C for P. gingivalis culture. Before P. gingivalis infection, the liquid medium was centrifuged (6,000 g, 10 min) and replaced by EBM‐2 medium without antibiotics.
Menadione
Manufactured by BD
Menadione is a synthetic form of vitamin K. It is a white to pale yellow crystalline powder with a molecular formula of C₆H₆O₂. Menadione is used as a dietary supplement and in the production of various pharmaceutical and laboratory products.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by BD (6 mentions)
Hemin, by BD (6 mentions)
Hemin, by Merck Group (5 mentions)
Cysteine, by BD (4 mentions)
Cysteine, by Fujifilm (4 mentions)
Menadione, by Merck Group (3 mentions)
Brain heart infusion, by BD (2 mentions)
Trypot soya agar, by Nissui Pharmaceutical (2 mentions)
L cysteine hydrochloride, by Fujifilm (1 mentions)
Menadione, by Nacalai Tesque (1 mentions)
Chloramphenicol, by Fujifilm (1 mentions)
Trypto soya agar, by Nissui Pharmaceutical (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hemin, by Thermo Fisher Scientific (1 mentions)
Tryptic soy agar, by BD (1 mentions)
Heart infusion broth, by BD (1 mentions)
Forma anaerobic chamber, by Thermo Fisher Scientific (1 mentions)
6 protocols using menadione
1
Cultivation of P. gingivalis for Infection Studies
2
Cultivation of Porphyromonas gingivalis Strains
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Bacterial strains used in this study are listed in Table 1 . P. gingivalis ATCC 33277 (wild-type) and sigP, sigCH, PGN_0450, PGN_0970, and sigH mutants were used. P. gingivalis strains were maintained anaerobically (10% CO2, 10% H2, and 80% N2) at 37°C on enriched tryptic soy (TS) agar (Becton Dickinson, Franklin Lakes, NJ) supplemented with 0.5% brain heart infusion (BHI) (Becton Dickinson), 0.1% cysteine (Wako Pure Chemical Industries, Osaka, Japan), 5 μg/mL hemin (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL menadione (Nacalai Tesque, Kyoto, Japan), and 5% defibrinated horse blood (Nippon Bio-Test Laboratories, Tokyo, Japan). When required, erythromycin (15 μg/mL) was added to the medium. For liquid cultures, P. gingivalis cells were grown in enriched BHI medium supplemented with 0.5% yeast extract (Becton Dickinson), 0.1% cysteine, 5 μg/mL hemin, and 0.5 μg/mL menadione [20 (link)].
3
Cultivation of P. gingivalis Strains
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P. gingivalis ATCC33277 and Lys gingipain mutant P. gingivalis KDP129 were used. P. gingivalis was maintained on blood agar plate containing 40 mg/ml trypto-soya agar (Nissui Pharmaceutical, Tokyo, Japan), 5 mg/ml brain heart infusion (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 1 g/ml cysteine (Wako Pure Chemical Industries, Osaka, Japan), 5 μg/ml hemin (Sigma-Aldrich, St. Louis, MO, USA), 1 μg/ml menadione (Sigma-Aldrich), 5% defibrinated sheep blood (Nippon Bio-test laboratories, Tokyo, Japan) in Bactron anaerobic chamber (Shel Lab, Cornelius, OR, USA) with 10% CO2, 10% H2, 80% N215 (link). KDP129 was maintained on blood agar plate with 20 μg/ml chloramphenicol (Wako Pure Chemical Industries). P. gingivalis was grown in enriched BHI broth containing 37 mg/ml brain heart infusion, 2.5 mg/ml yeast extract (Becton, Dickinson and Company), 1 g/ml cysteine, 5 μg/ml hemin and 1 μg/ml menadione. KDP129 was grown on BHI broth with 20 μg/ml chloramphenicol. Before P. gingivalis was cocultured with MG6 cells and primary cultured microglia, P. gingivalis culture medium was centrifuged (6000 × g, 10 min) and the supernatant was replaced with DMEM without FBS or penicillin-streptomycin.
4
Anaerobic Cultivation of Oral Pathogens
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T. forsythia ATCC 43037 cells were grown on agar plates containing 3.8% heart infusion broth (Becton Dickinson, Sparks, MD, USA), 0.005% N-acetylmuramic acid (Fisher Scientific, Pittsburgh, PA, USA), 5% fetal bovine serum (FBS) (Fisher Scientific), 5 µg/mL of hemin (Fisher Scientific), 0.5 µg/mL of menadione (Fisher Scientific), and 5% yeast extract (Becton Dickinson). Cells of P. gingivalis strain 381 were grown on blood agar plates containing 3% tryptic soy agar (Becton Dickinson,), 0.5% yeast extract, 5 µg/mL of hemin, 1 µg/mL of menadione, and 5% sheep’s blood. These bacteria were cultivated under an anaerobic atmosphere (10% H2, 5% CO2, and 85% N2) at 37°C in a Forma anaerobic chamber (Thermo Scientific, Waltham, MA, USA), as described elsewhere.
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T. forsythia ATCC 43037 cells were grown on agar plates containing 3.8% heart infusion broth (Becton Dickinson, Sparks, MD, USA), 0.005% N-acetylmuramic acid (Fisher Scientific, Pittsburgh, PA, USA), 5% fetal bovine serum (FBS) (Fisher Scientific), 5 µg/mL of hemin (Fisher Scientific), 0.5 µg/mL of menadione (Fisher Scientific), and 5% yeast extract (Becton Dickinson). Cells of P. gingivalis strain 381 were grown on blood agar plates containing 3% tryptic soy agar (Becton Dickinson,), 0.5% yeast extract, 5 µg/mL of hemin, 1 µg/mL of menadione, and 5% sheep’s blood. These bacteria were cultivated under an anaerobic atmosphere (10% H2, 5% CO2, and 85% N2) at 37°C in a Forma anaerobic chamber (Thermo Scientific, Waltham, MA, USA), as described elsewhere.
5
Culturing P. gingivalis for In Vitro and In Vivo Studies
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Porphyromonas gingivalis ATCC 33277 were cultured on blood BHI (brain heart infusion) agar plate containing 40 mg/ml trypot-soya agar (Nissui Pharmaceutical, Tokyo, Japan), 5 mg/ml BHI (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 1 g/ml cysteine( Wako Pure Chemical Industries, Osaka, Japan), 5 µg/ml hemin (Sigma-Aldrich, St. Louis, MO, USA), 1 µg/ml menadione (Sigma-Aldrich), 5% de brinated sheep blood (Nippon Bio-test laboratories, Tokyo, Japan) in bactron anaerobic chamber (Shel Lab, Cornelius, OR, USA) with mix gas of 10% CO 2 , 10% H 2 , 80% N 2 . P. gingivalis were grown in BHI medium containing 37 mg/ml BHI, 2.5 mg/ml yeast extract (Becton, Dickinson and Company), 1 g/ml cysteine, 5 µg/ml hemin and 1 µg/ml menadione. P. gingivalis were pelleted by centrifuge at 6000 × g and suspended by neurobasal before treatment in cell culture and suspended by 100µL PBS for vivo experiments.
6
Cultivation and Preparation of P. gingivalis
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Porphyromonas gingivalis ATCC 33277 were cultured on blood BHI (brain heart infusion) agar plate containing 40 mg/ml trypot-soya agar (Nissui Pharmaceutical, Tokyo, Japan), 5mg/ml BHI (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 1g/ml cysteine( Wako Pure Chemical Industries, Osaka, Japan), 5μg/ml hemin (Sigma-Aldrich, St. Louis, MO, USA), 1μg/ml menadione (Sigma-Aldrich), 5% defibrinated sheep blood (Nippon Bio-test laboratories, Tokyo, Japan) in bactron anaerobic chamber (Shel Lab, Cornelius, OR, USA) with mix gas of 10% CO2, 10% H2, 80% N2. P. gingivalis were grown in BHI medium containing 37 mg/ml BHI, 2.5 mg/ml yeast extract (Becton, Dickinson and Company), 1g/ml cysteine, 5μg/ml hemin and 1 μg/ml menadione. P.
gingivalis were pelleted by centrifuge at 6000×g and suspended by neurobasal before treatment in cell culture and suspended by 100μL PBS for vivo experiments.
gingivalis were pelleted by centrifuge at 6000×g and suspended by neurobasal before treatment in cell culture and suspended by 100μL PBS for vivo experiments.
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