A given pulverized frozen human fecal sample (353±184 mg; mean±SD) was transferred to an anaerobic Coy chamber (atmosphere 75% N2, 20% CO2, 5% H2) in a 2mL Axygen screw topped tube. The tube was then opened and its contents were transferred to a 50 mL conical shaped polypropylene tube (Falcon). The fecal material was suspended in 10 mL of sterile PBS supplemented with 0.1% L-cysteine (Sigma) by vortexing with sterile 2 mm-diameter glass beads. The suspension was passed through a nylon 100 μm mesh filter (BD) and the filtrate was mixed with an equal volume of 30% glycerol in PBS/0.1% cysteine. Aliquots (1.2 mL) of this suspension were placed amber glass vials, each of which was sealed with a crimp top, and frozen at −80°C. Tubes were thawed, and transferred into gnotobiotic isolators (with surface sterilization achieved by treatment with Clidox). Aliquots (200 μL) were then introduced into each germ-free mouse in a given experimental group by oral gavage. A total of 38 animals were used for this study (n=4–5 mice/donor microbiota). This size of each treatment group was not based on a formal power calculation but was informed by our previous work described in ref. 8 . There was no randomization of mice for this study; male C57BL/6J animals in each group were age- and weight-matched prior to gavage. Investigators were not blinded with respect to the donor microbiota.
Cysteine
Manufactured by BD
Cysteine is an amino acid that is commonly used in laboratory settings. It serves as a building block for proteins and plays a role in various biochemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by BD (5 mentions)
Cysteine, by Fujifilm (4 mentions)
Menadione, by BD (4 mentions)
Hemin, by BD (4 mentions)
Hemin, by Merck Group (4 mentions)
Menadione, by Merck Group (3 mentions)
Conical shaped polypropylene tube, by BD (2 mentions)
Nylon 100 μm mesh filter, by BD (2 mentions)
L cysteine, by Merck Group (2 mentions)
Trypot soya agar, by Nissui Pharmaceutical (2 mentions)
Cefoxitin, by BD (1 mentions)
Menadione, by Nacalai Tesque (1 mentions)
Chloramphenicol, by Fujifilm (1 mentions)
Brain heart infusion, by BD (1 mentions)
Trypto soya agar, by Nissui Pharmaceutical (1 mentions)
7 protocols using cysteine
1
Fecal Microbiome Transplant Protocol
2
Isolation and Typing of C. difficile from Fecal Samples
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Fecal samples were collected from patients previously identified as colonized or infected with C. difficile and plated anaerobically onto brain heart infusion (BHI) agar plates supplemented with yeast extract, cysteine, and the antibiotics cycloserine and cefoxitin (BHI and yeast extract were from BD Biosciences, and the other components were from Sigma-Aldrich). Individual colonies that were able to grow in the presence of these antibiotics and that had the characteristic phenotype of C. difficile were selected, isolated, and then typed using MLST (24 (link)).
3
Fecal Microbiome Transplant Protocol
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A given pulverized frozen human fecal sample (353±184 mg; mean±SD) was transferred to an anaerobic Coy chamber (atmosphere 75% N2, 20% CO2, 5% H2) in a 2mL Axygen screw topped tube. The tube was then opened and its contents were transferred to a 50 mL conical shaped polypropylene tube (Falcon). The fecal material was suspended in 10 mL of sterile PBS supplemented with 0.1% L-cysteine (Sigma) by vortexing with sterile 2 mm-diameter glass beads. The suspension was passed through a nylon 100 μm mesh filter (BD) and the filtrate was mixed with an equal volume of 30% glycerol in PBS/0.1% cysteine. Aliquots (1.2 mL) of this suspension were placed amber glass vials, each of which was sealed with a crimp top, and frozen at −80°C. Tubes were thawed, and transferred into gnotobiotic isolators (with surface sterilization achieved by treatment with Clidox). Aliquots (200 μL) were then introduced into each germ-free mouse in a given experimental group by oral gavage. A total of 38 animals were used for this study (n=4–5 mice/donor microbiota). This size of each treatment group was not based on a formal power calculation but was informed by our previous work described in ref. 8 . There was no randomization of mice for this study; male C57BL/6J animals in each group were age- and weight-matched prior to gavage. Investigators were not blinded with respect to the donor microbiota.
4
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)].
5
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.
6
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.
7
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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