Before analysis, the rinds were removed from the cheeses. For LAB counts, 5 g of cheese was added to 50 mL 2% sodium citrate and homogenized using IKA T25 digital Ultra-Trrax (12,000 rpm, 1 min at room temperature; IKA Werke GmbH and Co. KG). Each homogenized sample was spread onto an M17 (Difco Laboratories) agar plate supplemented with 0.5% glucose. Plates were incubated at 30°C in an AnaeroPack system (AnaeroPack, Mitsubishi Gas Chemical). For pH analysis, 5 g of cheese was added to 10 mL sterilized distilled water and homogenized. After centrifugation at 12,000 rpm for 10 min, the pH of the supernatant was analyzed using a pH meter (Seven Easy pH, Mettler Toledo). Differences between cheese supplemented with sake lees (n = 3) and control cheese (n = 3) were assessed using t-test. A P-value <0.05 was considered to indicate statistical significance.
Anaeropack
Manufactured by Mitsubishi
Sourced in Japan, United States
AnaeroPack is a compact laboratory equipment designed for creating anaerobic conditions. It generates an anaerobic atmosphere within enclosed spaces, such as jars or pouches, without the use of external gas supply. The device functions through a chemical reaction that consumes oxygen and produces carbon dioxide, thus establishing an anaerobic environment suitable for various microbiological applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Anaer indicator, by bioMérieux (3 mentions)
Rs 2000 pro x ray bio irradiator, by Rad Source (3 mentions)
Eca109, by American Type Culture Collection (3 mentions)
Icg 001, by Apexbio (2 mentions)
Gifu anaerobic medium broth, by Nissui Pharmaceutical (2 mentions)
Gam broth, by Nissui Pharmaceutical (2 mentions)
Earle s balanced salt solution, by Leagene (2 mentions)
Anaerobic chamber, by Coy Laboratory Products (2 mentions)
L cysteine, by Kanto Chemical (2 mentions)
Bhi broth, by BD (2 mentions)
Aneropack anaero, by Mitsubishi (2 mentions)
Gifu anaerobic medium, by Nissui Pharmaceutical (2 mentions)
Anaeropack anaero, by Mitsubishi (2 mentions)
Carboxymethylcellulose, by Merck Group (2 mentions)
Anaerobic jar, by BD (2 mentions)
Modified gifu anaerobic medium broth, by Nissui Pharmaceutical (2 mentions)
Bolton broth, by Thermo Fisher Scientific (2 mentions)
127 protocols using anaeropack
1
Lactic Acid Bacteria and pH Analysis
2
Isolation of O. oeni and L. plantarum from Pinot noir Wine
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O. oeni and L. plantarum isolates were obtained from Pinot noir wine samples from a 2012 vintage, in which alcoholic fermentation (AF) and MLF were spontaneous. At the end of MLF the wine had the following values: pH 3.75, 14.3% (v/v) ethanol, L-malic acid 0.5 g/L.
Samples were aseptically collected from a commercial cellar in General Roca, Argentinean North Patagonia, and inoculated in MLO (Maicas et al. 1999) and MRS (Biokar Diagnostic, Beauvais, France) (De Man et al. 1960 ) plates supplemented with cycloheximide 100 mg/L, under anaerobic conditions (AnaeroPack -Mitsubishi Gas Chemical America, Inc., New York, NY), at 28 °C, during 7 days or 48 h, respectively.
Isolates identified as O. oeni or L. plantarum were grown in MLO or MRS broth, respectively. Cultures were kept frozen at -20 °C in the corresponding broth supplemented with glycerol (30% v/v).
Samples were aseptically collected from a commercial cellar in General Roca, Argentinean North Patagonia, and inoculated in MLO (Maicas et al. 1999) and MRS (Biokar Diagnostic, Beauvais, France) (De Man et al. 1960 ) plates supplemented with cycloheximide 100 mg/L, under anaerobic conditions (AnaeroPack -Mitsubishi Gas Chemical America, Inc., New York, NY), at 28 °C, during 7 days or 48 h, respectively.
Isolates identified as O. oeni or L. plantarum were grown in MLO or MRS broth, respectively. Cultures were kept frozen at -20 °C in the corresponding broth supplemented with glycerol (30% v/v).
3
Isolation and Cultivation of Sulfate-Reducing Bacteria
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In a portable glove box filled with N2 (Captairpyr, Erlab Company, Val-de-Reuil, France), which ensured that no oxygen was present in the operating environment, the prepared bacterial suspension was spread on Postgate’s B (PGB) modified medium (anaerobic medium for SRB) plates [30 ,34 ] containing KH2PO4 (0.5 g·L−1), NH4Cl (1.0 g·L−1), Na2SO4 (1.0 g·L−1), CaCl2·2H2O (0.1 g·L−1), MgSO4·7H2O (2.0 g·L−1), C3H5O3Na (80%) (3.5 mL·L−1), yeast extract (1.0 g·L−1), FeSO4·7H2O (0.5 g·L−1), agar (20.0 g·L−1), vitamin C (0.1 g·L−1), and H-Cys-OH·HCl (0.5 g·L−1) with a pH between 7.0 and 7.2. These plates were sealed and put into 2.5 L round-bottomed vertical anaerobic culture bags with AnaeroPack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) and then cultured at 25 °C for 72 h. When the medium turned black, this indicated the presence of SRB. Then, plates of a suitable dilution were selected, and the colonies grown on them were isolated and purified by streaking several times to obtain single colonies that could darken the medium [35 (link)]. Those strains that were able to blacken the medium and had a rotten egg smell were considered to be SRB [36 (link)] and were cultured in anaerobic Postgate’s B (PGB) modified liquid medium in penicillin bottles [30 ] to carry out subsequent research.
4
Modulating S100A16 Expression in NRK-49F Cells
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Normal NRK-49F cells were cultured in DMEM/F12 (Gibco, California, USA) containing 10% fetal bovine serum (BI, Israel) at 37 o C in 5% CO 2 /95% air. NRK-49F cells were the gifts from Dr. Chunsun Dai (Center for Kidney Diseases, 2nd A liated Hospital, Nanjing Medical University). NRK-49F cells were pretreated with different concentrations of Wnt/β-catenin signaling pathway small molecule inhibitor ICG-001 (APExBIO, Houston, USA) under hypoxia/reoxygenation. Hypoxia 24 hours and reoxygenation 24 hours in NRK-49F cells were established with Anaeropack (Mitsubishi gas chemical company, Japan).
Transient transfections of plasmids, shRNA and adenovirus
The constructs of S100A16 OE, pcDNA3.1 (control plasmids of S100A16 OE), S100A16 shRNA, and scrambled shRNA (control S100A16 shRNA), were the gifts of Dr. Yun Liu (The First A liated Hospital of Nanjing Medical University) [25] (link). NRK-49F cells pretreated by ICG-001 were transiently transfected with S100A16 OE or pcDNA3.1 by using lipofectamine 3000 reagent (Invitrogen, CA, USA). Transfection of S100A16 shRNA or scrambled shRNA using lipofectamine 3000 reagent down-regulated the expression of S100A16 in NRK-49F cells, and then NRK-49F cells were collected and subjected to different analyses. HRD1 adenovirus was obtained from Shanghai Genechem Co.
Transient transfections of plasmids, shRNA and adenovirus
The constructs of S100A16 OE, pcDNA3.1 (control plasmids of S100A16 OE), S100A16 shRNA, and scrambled shRNA (control S100A16 shRNA), were the gifts of Dr. Yun Liu (The First A liated Hospital of Nanjing Medical University) [25] (link). NRK-49F cells pretreated by ICG-001 were transiently transfected with S100A16 OE or pcDNA3.1 by using lipofectamine 3000 reagent (Invitrogen, CA, USA). Transfection of S100A16 shRNA or scrambled shRNA using lipofectamine 3000 reagent down-regulated the expression of S100A16 in NRK-49F cells, and then NRK-49F cells were collected and subjected to different analyses. HRD1 adenovirus was obtained from Shanghai Genechem Co.
5
Measuring Cholesterol Release from H. pylori
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A bacterial suspension (200 μl) of H. pylori strain NCTC 11638 was cultured for 24 h with shaking in a PPLO broth (10 ml) containing a 30 μM concentration of FC and 10 μM concentration of 2,6-di-O-methyl-β-cyclodextrin (dMβCD: Sigma-Aldrich Inc.) in the microaerophilic chamber. This culture procedure was repeated three times to prepare the FC-retaining H. pylori cells. After three washes with PBS by centrifugation (10000 × g, 5 min), the bacterial cells (109 CFU) were suspended in PBS (5 ml) containing dMβCD (30 μM) and shaken for 48 h in the presence or absence of VDP1 (10 μg/ml) at 37°C in a box filled with anaerobic gas using an AnaeroPack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). The CFUs were counted, the OD660 nm of the bacterial suspension (200 μl) was measured and the bacterial cells stained with CBB were observed. After filtrating the bacterial suspension (4 ml) through a GDXS 25 syringe filter (Whatman, Buckinghamshire, UK), the lipids from the H. pylori cells were extracted from the filtrates by the organic solvent distribution method to measure the FC amounts released from the cell membranes.
6
Anaerobic Culture of S. gordonii
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S. gordonii ATCC10558 [23 (link)] was grown at 37 °C anaerobically with AnaeroPack (Mitsubishi Gas Chemical, Tokyo, Japan) in Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) and used for downstream assays as described below.
7
Formation and Analysis of P. acnes Biofilm
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A biofilm of P. acnes JK12.2 was formed after anaerobic cultivation in 2 ml of GAMG Broth using a 12-well flat-bottomed polystyrene plate in an air-tight container containing an Anaero-Pack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at 37°C for 3 days. Biofilms were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature and then with 1% osmic acid in phosphate buffer at 4°C for 1 h. Preparation of thin sections, staining, and microscopic observation were performed as described previously (Sugimoto et al., 2016 (link)).
8
Anaerobic Cultivation of Cardiac Pacemaker Isolates
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Cardiac pacemaker devices were removed in the operation room, placed on sterilized steel containers, and stamped on Anaero Columbia Agar with Rabbit Blood (Becton Dickinson, Franklin Lakes, NJ, United States) containing 2.5% rabbit blood. They were subsequently incubated anaerobically in an air-tight container with an Anaero-Pack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at 37°C for 7 days. Single-colony isolates were cultured anaerobically in Gifu Anaerobic Media (GAM) Broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) at 37°C for 3 days and stocked at -80°C.
9
Quantification of P. acnes Biofilm Formation
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Propionibacterium acnes isolates were cultured anaerobically in GAM Broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) in an air-tight container containing an Anaero-Pack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at 37°C for 3 days. Cultures were diluted to an OD595 of 0.01 (corresponding to 2 × 107 colony-forming units/ml) with fresh GAM Broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 1% glucose (GAMG Broth). Aliquots (200 μl) of this bacterial suspension were added to the wells of a 96-well flat-bottomed polystyrene plate and incubated anaerobically for 3 days at 37°C. Cell growth of P. acnes isolates was evaluated by measuring the absorbance at 595 nm. After removal of the supernatants, biofilms formed on the bottom of the wells were stained with 0.1% crystal violet for 10 min and subsequently washed twice with 200 μl of phosphate-buffered saline (PBS). Next, 200 μl of ethanol was added to the wells to extract crystal violet and the absorbance at 595 nm was measured using an Infinite 200 PRO Microplate Reader (Tecan, Mannedorf, Switzerland).
10
Evaluating HUVEC Angiogenic Potential
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The formation of vascular-like structures by HUVECs on growth factor-reduced Matrigel (BD Biosciences) was performed as previously described (20,27). Differentiation was quantified by measuring the area of the “tube-like” networks that form in three randomly chosen fields from each well. Each experiment was repeated three times. Chemotaxis of HUVECs was assessed by transwell assay with polycarbonate membranes coated with fibronectin (Corning)(3415) [25 (link)]. HUVECs were added to the upper chamber, and serum-deprived media supplemented with pemafibrate, FGF21 or vehicle was added to the lower chamber. Cells were allowed to migrate through the pores of the membrane for 10 hours. Cell proliferation was assessed by MTS-based assay (Promega)(G3580) [26 (link)]. HUVECs were stimulated with pemafibrate, FGF21 or vehicle for the indicated lengths of time under normoxic or hypoxic condition. Hypoxic conditions were generated using an AnaeroPack (5% O2, 5% CO2, Mitsubishi GAS Chemical)(3276LJ).
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