E. multilocularis primary cells and metacestode vesicles were isolated, separated from host contaminants and maintained in axenic cultures as previously described [40] (link). For the collection of E/S products, axenically maintained parasite larvae were washed thrice in 1× PBS and resuspended in collection medium DMEM10redox; i.e. dulbecco's modified eagle's medium, 4.5 g glucose/L (Gibco) supplemented with 10% fetal bovine serum Superior (Biochrom AG), 100 µg/ml penicillin;streptomycin (Biochrom AG), 20 µg/ml levofloxacin (Sanofi-Aventis), 143 µM ß-mercapthoethanol (Sigma-Aldrich), 10 µM bathocuproine disulfonic acid (Sigma) and 100 µM L-cysteine (Sigma) under axenic conditions (i.e. sealed in Nitrogen filled Ziploc freezer bag and placed in a 5% CO2 incubator at 37°C). After 48 hours of culture, the supernatants containing the larval E/S products were collected and filtered through a 0.2 µm sieve (Filtropur S filter, SARSTEDT). The total amount of proteins per supernatant was determined using the bicinchoninic acid assay (ThermoScientific) and normalized by addition of ddH2O prior to storage at −80°C until use.
Filtropur s filter
Manufactured by Sarstedt
Sourced in Germany
Filtropur S filters are membrane filters designed for laboratory use. They are made of cellulose acetate and have a pore size of 0.2 microns. The filters are suitable for the filtration of aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
L cysteine, by Merck Group (2 mentions)
Fetal bovine serum superior, by Harvard Bioscience (2 mentions)
Bathocuproinedisulfonic acid, by Merck Group (2 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Levofloxacin, by Sanofi (1 mentions)
Sorvall lynx 4000, by Thermo Fisher Scientific (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
β mercapthoethanol, by Merck Group (1 mentions)
Levofloxacin tavanic, by Sanofi (1 mentions)
T75 flask, by Greiner (1 mentions)
Nutrient broth, by Thermo Fisher Scientific (1 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
11 protocols using filtropur s filter
1
Isolation and Culture of E. multilocularis Primary Cells
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E. multilocularis primary cells and metacestode vesicles were isolated, separated from host contaminants and maintained in axenic cultures as previously described [40] (link). For the collection of E/S products, axenically maintained parasite larvae were washed thrice in 1× PBS and resuspended in collection medium DMEM10redox; i.e. dulbecco's modified eagle's medium, 4.5 g glucose/L (Gibco) supplemented with 10% fetal bovine serum Superior (Biochrom AG), 100 µg/ml penicillin;streptomycin (Biochrom AG), 20 µg/ml levofloxacin (Sanofi-Aventis), 143 µM ß-mercapthoethanol (Sigma-Aldrich), 10 µM bathocuproine disulfonic acid (Sigma) and 100 µM L-cysteine (Sigma) under axenic conditions (i.e. sealed in Nitrogen filled Ziploc freezer bag and placed in a 5% CO2 incubator at 37°C). After 48 hours of culture, the supernatants containing the larval E/S products were collected and filtered through a 0.2 µm sieve (Filtropur S filter, SARSTEDT). The total amount of proteins per supernatant was determined using the bicinchoninic acid assay (ThermoScientific) and normalized by addition of ddH2O prior to storage at −80°C until use.
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E. multilocularis primary cells and metacestode vesicles were isolated, separated from host contaminants and maintained in axenic cultures as previously described [40] (link). For the collection of E/S products, axenically maintained parasite larvae were washed thrice in 1× PBS and resuspended in collection medium DMEM10redox; i.e. dulbecco's modified eagle's medium, 4.5 g glucose/L (Gibco) supplemented with 10% fetal bovine serum Superior (Biochrom AG), 100 µg/ml penicillin;streptomycin (Biochrom AG), 20 µg/ml levofloxacin (Sanofi-Aventis), 143 µM ß-mercapthoethanol (Sigma-Aldrich), 10 µM bathocuproine disulfonic acid (Sigma) and 100 µM L-cysteine (Sigma) under axenic conditions (i.e. sealed in Nitrogen filled Ziploc freezer bag and placed in a 5% CO2 incubator at 37°C). After 48 hours of culture, the supernatants containing the larval E/S products were collected and filtered through a 0.2 µm sieve (Filtropur S filter, SARSTEDT). The total amount of proteins per supernatant was determined using the bicinchoninic acid assay (ThermoScientific) and normalized by addition of ddH2O prior to storage at −80°C until use.
2
Pyoverdine Extraction from Bacterial Culture
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Pyoverdines or precursors were extracted based on the method by Meyer et al. (37 (link)). Briefly, 1 ml of an overnight culture (in CAA medium) was used to inoculate 1 l CAA medium. The culture was grown for 72 h (30 °C, 180 rpm), cells were sedimented (30′, 20,000g, 4 °C; Sorvall Lynx 4000 centrifuge; Thermo Fisher Scientific), and the supernatant was sterile filtered (0.2 μm pore size) using the Filtropur V50 system (Sarstedt, Inc). The pH was adjusted to 6.0 using 8 M HCl, and 20 g/l XAD-4 resin was added. After 3 h incubation at 4 °C, the resin was filtered, the flow through was discarded, and the resin was resuspended in 0.5 l H2O. After another incubation for 1 h at 4 °C, the resin was filtered, resuspended in 200 ml 15% methanol, incubated for 15 min at 4°, and filtered again. Then, the resin was resuspended in 150 ml 50% methanol, incubated for 1 h at 4°, and filtered. The solvent was removed using a rotary evaporator, kept at 30 °C (Rotavapor R-124), and the pyoverdine or its biosynthetic intermediates were resuspended in 1 ml H2O and filtered (Filtropur S filter; Sarstedt, Inc).
3
EBV Transformation of Primary Human B Cells
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To prolong the viability of primary human B cells (n = 4 anti‐MAG neuropathy patients) ex vivo, the cells were transformed with Epstein‐Barr virus (EBV) (Hollyoake, Stühler, Stühler, Farrell, Gordon, & Sinclair, 1995 ). Marmoset B95.8 Epstein–Barr virus‐producing cells (RRID:CVCL_1953; Vircell) were maintained in complete RPMI 1640 medium containing 10% FBS, 1% AA, and 1% l ‐Gln. The supernatant containing the EBV was collected after 20 days by centrifuging cells (1400g, 15 min) and filtering the supernatant with a 0.45 μm and a 0.2 μm Filtropur S filter (83.1826; Sarstedt). Freshly isolated human PBMC (1x106 cells) were resuspended in 3 ml EBV supernatant and incubated for 24 hr before adding 7 ml complete RPMI 1640 medium supplemented with 20 ng/ml cyclosporin A (30024; Sigma‐Aldrich). The cell growth was monitored and the medium changed every 2–3 days to achieve an efficient immortalization. After 14 days of culture, cells were maintained in complete RPMI 1640 medium without cyclosporin A, at a density of approximately 5 × 105 to 1 × 106 cells/mL.
4
Isolation and Cultivation of Echinococcus multilocularis Metacestodes
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Echinococcus multilocularis metacestodes were isolated, separated from host contaminants and axenically cultivated as previously described (6 (link)). For the collection of E/S products, metacestode vesicles were kept under axenic conditions for 10 days, washed three times in 1 x PBS and resuspended in DMEM10redox i.e., DMEM + GlutamaxTM, GIBCO supplemented with 10% Fetal Bovine Serum Superior (Biochrom AG), 100 μg/ml penicillin/streptomycin (PenStrep solution, Biochrom AG), 20 μg/ml Levofloxacin (Tavanic, Sanofi-Aventis), β-mercapthoethanol (143 μM, Sigma-Aldrich, cat. M6250), 10 μM Bathocuproine disulfonic acid (Sigma, cat. B-1125) and 100 μM L-Cysteine (Sigma, cat. C-1276) under axenic conditions [see (6 (link)) for description of conditions]. After 48 h of culture, the supernatants containing the MVE/S were collected and filtered through a 0.2 μm sieve (Filtropur S filter, SARSTEDT). The total amount of E/S product proteins was determined as previously defined (6 (link)) and the E/S products stored at −80°C until use.
5
Cell Seeding and Supernatant Filtration
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Cells were seeded in a density of 5000 cell/cm2 in T75 flasks (Greiner Bio-One, Kremsmünster, Austria) in 12 ml cultivation medium and incubated for 72 h at 37 °C and 5% CO2. The supernatant was sterile filtered with a Filtropur S filter (Sarstedt, Nümbrecht, Germany) with a pore size of 0.2 µm and used immediately.
6
Bacterial Culture Filtrate Extraction
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Liquid cultures of B. velezensis, strain QST 713 and Kos were grown in 50 ml nutrient broth (NB) (Oxoid) at 30 °C, 120 rpm, in the dark in an orbital incubator. At various time points (24, 48, 72 and 96 h), flasks were removed from the incubator and the culture filtrate (CF) from the bacterial cultures were collected through centrifugation (1000 x g, 20 min). CF was filtered through a 0.45 μm filtropur S filters (Sarstedt Ltd). CF stocks from the bacterial strains were kept frozen at −20 °C until required.
7
Analyzing Gliotoxin Production in A. fumigatus
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A. fumigatus (ATCC 26933) and a gliotoxin-deficient strain of A. fumigatus Af293 (ΔgliZ) were grown and maintained on Sabouraud dextrose agar (Oxoid) at 37 °C. Conidia were harvested using 0.01% Tween-80 and washed twice in phosphate buffered saline (PBS). Conidial number was ascertained by hemocytometry and conidia were added to Czapek-Dox liquid media (Duchefa) at a final concentration of 5 × 105/ml. The cultures were incubated for 24, 48, 72, and 96 h at 37 °C in an orbital incubator, at 200 rpm. After each time point, the hyphal mass was harvested, and the wet weight was recorded. The A. fumigatus culture-filtrates (CuF) were filter-sterilized using 0.2 μm filtropur S filters (Sarstedt).
8
Cultivation and Filtration of P. aeruginosa
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P. aeruginosa was cultivated on nutrient agar (Oxoid) at 37 °C. A sterile loop was used to inoculate Czapek-Dox liquid media with the bacteria. Bacterial cultures were incubated for 48 h at 37 °C in an orbital incubator, at 200 rpm. The concentration of the bacterial suspension was quantified by obtaining the optical density at 600 nm (OD600). Bacterial densities were measured after 48 h incubation (OD 0.19 ± 0.022). Bacteria were separated from the supernatant by centrifugation (15 min, 2000 g). P. aeruginosa culture filtrates were sterilized using 0.2 μm filtropur S filters (Sarstedt) and are hereafter referred to as P. aeruginosa CuF.
9
Fungal-Bacterial Co-culture Interaction Dynamics
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A. fumigatus conidia (5 × 105 conidia/ml) and P. aeruginosa (loopful) were cultured in Czapek-Dox liquid media for 24 h at 37 °C in an orbital incubator at 200 rpm. Prior to co-incubation with A. fumigatus cultures, P. aeruginosa density was determined to ensure similar densities across the groups (OD600 0.2 ± 0.0087). The co-culture was incubated under the same conditions for a further 24 h to give a total incubation period of 48 h for each pathogen to match the total incubation time applied for the fungal- and bacterial-only cultures. The hyphae were removed from the culture and the bacteria were removed by centrifugation (20 min at 2000 g). The Co-culture culture filtrates were sterilized using 0.2 μm filtropur S filters (Sarstedt) and are hereafter referred to as Co-culture CuF. A. fumigatus CuF, P. aeruginosa CuF and Co-culture CuF were also produced as described above in minimal media (MM) and synthetic cystic fibrosis medium (SCFM) (21 (link)).
10
Cultivation and Filtration of A. fumigatus
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A. fumigatus conidia (5×105 ml−1) were added to Czapek-Dox liquid media (pH 6.8). The cultures were incubated for 48 h at 37 °C in an orbital incubator at 200 r.p.m. The hyphal mass was harvested and the wet weight was recorded. The A. fumigatus CuF was sterilized using 0.2 µm filtropur S filters (Sarstedt) and the pH was measured (4.5).
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A. fumigatus conidia (5×105 ml−1) were added to Czapek-Dox liquid media (pH 6.8). The cultures were incubated for 48 h at 37 °C in an orbital incubator at 200 r.p.m. The hyphal mass was harvested and the wet weight was recorded. The A. fumigatus CuF was sterilized using 0.2 µm filtropur S filters (Sarstedt) and the pH was measured (4.5).
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