Co-aggregation study was evaluated as follows: 129AX-1 tubes with slides (SterilinTM, Thermo Fisher Scientific Inc., Rodano (MI), Italy) were added with 500 µL of an overnight culture of Lactobacillus strains grown at 37 °C in MRS broth (Oxoid, Thermo Fisher Scientific Inc., Rodano (MI), Italy, CM0359) under micro-aerobic conditions and 500 µL of an overnight culture of Candida spp. grown at 33 °C in DMEM (Dulbecco’s MEM—Biochrom GmbH, Berlin, Germany). Tubes were incubated at 37 °C for 4 h. Then, Gram staining was used to visualize the co-aggregates under oil immersion microscopy (1000×) [30 (link)]. Scoring was done according to Reid et al. [31 (link)]. Moreover, the co-aggregation were also evaluated on Hep-2 and Caco-2 cells. Tubes with monolayer cells were incubated at 37 °C for 4 h after inoculation with Candida and Lactobacillus strains, then Giemsa staining was used to visualize co-aggregates and adhesion on cells.
Dulbecco s mem
Manufactured by Harvard Bioscience
Sourced in Germany
Dulbecco's Modified Eagle's Medium (DMEM) is a cell culture medium used to support the growth of various cell types in vitro. It provides a balanced salt solution with added nutrients, vitamins, and other components necessary for cell proliferation and maintenance.
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Lab products found in correlation
Falcon cultureslides, by Corning (3 mentions)
Skbr3 cell line, by Cytion (2 mentions)
Sterilintm, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum fbs, by PromoCell (1 mentions)
Hek blue null1, by InvivoGen (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Fucus vesiculosus, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytion (1 mentions)
Dmem hams f12, by Cytion (1 mentions)
L glutamine, by Cytion (1 mentions)
Calcium chloride dehydrate, by Merck Group (1 mentions)
Potassium chloride (kcl), by Avantor (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Dimethyl sulfoxide (dmso), by Avantor (1 mentions)
Sodium hydroxide (naoh), by Avantor (1 mentions)
Fbs superior, by Harvard Bioscience (1 mentions)
Trypsin edta solution, by Harvard Bioscience (1 mentions)
D luciferin beetle monosodium salt, by Promega (1 mentions)
8 protocols using dulbecco s mem
1
Co-aggregation of Lactobacillus and Candida
2
Transfection and Coincubation Assays
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HEK293-T, HeLa or HEK-Blue Null1 cells (#hkb-null1, Invivogen) were used for transfection and coincubation assays and propagated in Dulbecco’s MEM (Biochrom, Berlin, Germany) supplemented with 10% [v/v] fetal bovine serum (FBS; Promocell). Growth medium of HEK-Blue Null1 cells was additionally supplemented with 100 µg/ml of zeocin as selective antibiotic. HD-11 chicken macrophage-like cell line used for nucleofection experiments was maintained in Iscove’s basal medium (IBM) supplemented with 10% [v/v] FBS. HD-11 cells stably transfected with the firefly luciferase gene under control of a NF-κB promoter were cultured in medium containing puromycin as selective antibiotic (5 g/liter). All cell lines were routinely kept at 37 °C in a 5% CO2 humidified atmosphere.
3
Culture and Maintenance of SKBR3 and HaCaT Cells
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SKBR3 cells were purchased from Cell Line Service (Eppelheim, Germany). The cells were cultured in Dulbecco’s MEM (Biochrom, Berlin, Germany) containing 10% fetal calf serum and 1% penicillin/streptomycin in a humidified chamber at 37°C and 5% CO2. The medium renewal was performed every 2–3 days. Cell harvesting was performed at a confluence of 70–80% with Accutase. The SKBR3 cells were seeded in culture slides (30,000 cells per well; Falcon CultureSlides, Corning Life Science, NY, USA, Cat.# 354108) or used as indicated for other experiments.
The HaCaT cells were obtained and handled as described in previous publications of our group [13 (link)]. Cells were harvested with Trypsin in a humidified chamber at 37°C and 5% CO2. Subsequently, the cells were seeded on culture slides (Falcon CultureSlides, Corning Life Science, Cat.# 354108) or 96-well plates (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany, Cat.# CLS3340).
The HaCaT cells were obtained and handled as described in previous publications of our group [13 (link)]. Cells were harvested with Trypsin in a humidified chamber at 37°C and 5% CO2. Subsequently, the cells were seeded on culture slides (Falcon CultureSlides, Corning Life Science, Cat.# 354108) or 96-well plates (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany, Cat.# CLS3340).
4
SKBR3 Cell Culturing Protocol
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We obtained the SKBR3 cell line from Cell Line Service (Eppelheim, Germany). The cells were cultured in Dulbecco´s MEM (Biochrom) supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin at 37°C and 5% CO2 in a humidified chamber. The medium was renewed every 2–3 days. They were cultured until 70–80% confluence. Cell harvesting was performed with Accutase treatment for 10 min in a humidified chamber at 37°C and 5% CO2. Subsequently, the cells were rinsed with sterile phosphate buffered saline (PBS) and counted using a Neubauer counting chamber. The cells were seeded in a density of 30.000–40.000 per well in culture slides (Falcon CultureSlides, Corning Life Science, New York, USA, Cat.# 354108).
5
Fucoidan Effects on Osteosarcoma and OECs
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MG63 osteosarcoma cells were cultivated in Dulbecco’s MEM (Biochrom, Berlin, Germany) supplemented with 10% FBS, 1% l -Glutamine (Gibco, Darmstadt, Germany) and 1% Pen/Strep. Cell subculture was performed after reaching 80–90% confluence. MG63 cells with different passage numbers within the range from 15 to 23 were used in mono-cultures and co-cultures with OECs.
For mono-cultures of MSC, MG63 or OEC, cells were seeded in fibronectin (Millipore, Temecula, CA, USA) coated 24 well-plates at a density of 40.000 cells/cm2. After one day, growth medium with different concentrations fucoidan from fucus vesiculosus (Sigma-Aldrich, Taufkirchen, Germany) was applied to the growth medium or cells were maintained in normal growth medium as controls.
For co-cultures the seeding of MSC and MG63 was performed as described above followed by adding OEC on the next day at a density of 40.000 cells/cm2. EGM-2 medium supplemented with 100 μg/mL fucoidan was applied to co-cultures one day after seeding of OECs, or cells were grown in EGM-2 as controls. Medium changes were performed every 3 days for both mono- and co-cultures.
For mono-cultures of MSC, MG63 or OEC, cells were seeded in fibronectin (Millipore, Temecula, CA, USA) coated 24 well-plates at a density of 40.000 cells/cm2. After one day, growth medium with different concentrations fucoidan from fucus vesiculosus (Sigma-Aldrich, Taufkirchen, Germany) was applied to the growth medium or cells were maintained in normal growth medium as controls.
For co-cultures the seeding of MSC and MG63 was performed as described above followed by adding OEC on the next day at a density of 40.000 cells/cm2. EGM-2 medium supplemented with 100 μg/mL fucoidan was applied to co-cultures one day after seeding of OECs, or cells were grown in EGM-2 as controls. Medium changes were performed every 3 days for both mono- and co-cultures.
6
Culturing SK-BR3 and BT474 Breast Cancer Cells
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We obtained the SK-BR3 cell line and the BT474 cell line from Cell Line Service (Eppelheim, Germany). The SK-BR3 cells were cultured in Dulbecco's MEM (Biochrom) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C and 5% CO2 in a humidified chamber. BT474 cells were cultured in DMEM:Hams F12 (1:1) supplemented with L-glutamine, Insulin and FBS (Cell Lines Service, Eppelheim, Germany). The medium was renewed every 2–3 days. They were cultured until 70–80% confluence. Cell harvesting was performed with Accutase treatment for 10 min in a humidified chamber at 37 °C and 5% CO2. Subsequently, the cells were rinsed with sterile phosphate-buffered saline (PBS) and counted using a Neubauer counting chamber. The cells were seeded at a density of 30,000–40,000 per well in culture slides (Falcon CultureSlides, Corning Life Science, New York, USA, Cat.# 354108). Mycoplasm infection was excluded with 4′,6-diamidino-2-phenylindole (DAPI) staining for each test.
7
Pyrazine-based Odor Compound Screening
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Dulbecco´s MEM (#F0435), FBS superior (#S0615), Lglutamine (#K0282), penicillin (10 000 U/mL)/streptomycin (10 000 U/mL) (#A2212), trypsin/EDTA solution (#L2143) (formerly Biochrom, Berlin, Germany, now Merck KGaA), calcium chloride dehydrate (#22322.295), D-glucose (#101174Y), dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride (#26764.230), sodium hydroxide (#28244.295) (VWR International GmbH, Darmstadt, Germany), sodium chloride (#1064041000, Merck KGaA, Darmstadt, Germany), and D-luciferin (beetle) monosodium salt (#E464X, Promega, Madison, USA), 2,5-dihydro-2,4,5-trimethylthiazoline (CAS# 4145-93-1) (#AB494350, abcr GmbH, Karlsruhe, Germany).
Pyrazines and other odorants are listed in Table S1. Compounds of particular interest for our experiments are listed alongside their assigned compound numbers, CAS numbers, structures, acronyms, and designation as KFO and/ or semiochemical in Table 1. Compounds utilized in the KFO screen were as previously published 38, 39 (Table S2).
Pyrazines and other odorants are listed in Table S1. Compounds of particular interest for our experiments are listed alongside their assigned compound numbers, CAS numbers, structures, acronyms, and designation as KFO and/ or semiochemical in Table 1. Compounds utilized in the KFO screen were as previously published 38, 39 (Table S2).
8
Immortalized Human Corneal Endothelial Cells
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Cell cultivation SV40 immortalized human corneal endothelial cells from a 91-year-old Caucasian woman, initially generated by Bednarz et al. (2000) ; Nitschke et al. (2007); Valtink et al. (2008) , were acquired from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (HCEC-12, Braunschweig, Germany). They were cultured in medium (M1), consisting of 95% DMEM (Dulbecco's MEM, Biochrom GmbH, Berlin, Germany) supplemented with 5% FBS (Fetal Bovine Serum, GE Healthcare, Freiburg, Germany), 1/100 Penicillin-Streptomycin (Sigma-Aldrich, Munich, Germany) and 1/125 Amphotericin B (Sigma-Aldrich), in T75 culture flasks (Sigma-Aldrich) at 37 °C in an incubator with 5% CO 2 (BBD 622, Thermo Scientific, Waltham, MA, USA). They were passaged every 7 days using 1 ml Trypsin-EDTA (0.05%; Gibco Invitrogen, Darmstadt, Germany) for 3 min. Trypsin activity was interrupted by adding 5 ml of M1. Cells were diluted in a 1:10 ratio.
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