Synechococcus strains were grown in A+ medium (37 ) containing sodium nitrate (1 g/l), supplemented with zeocin at 100 µg/ml where appropriate. Plates of solid A+ media were made following the addition of 1% agarose and 1 mM sodium thiosulphate. Cultures were grown photoautotrophically in 40 ml of liquid medium in 250 ml baffled flasks under continuous white LED illumination in an Algaetron growth chamber (PSI Instruments) at 200 µmol photons m−2 s−1 at 37°C with shaking at 200 rpm. Transformation plates were incubated at lower irradiances in a Multitron incubator (Infors) with continuous cool white fluorescent illumination at 50–70 µmol photons m−2 s−1 at 30°C. Light irradiance was measured using a Li-Cor Li-250A light sensor equipped with a Li-190SA quantum sensor. To generate growth profiles for the various strains, cells were cultured in 25 cm2 vented Corning tissue-culture flasks and the cell density was determined every 24 h for 7 days by measuring the OD740nm using a Fluostar Optima microplate reader (BMG Labtech).
Multitron incubator
Manufactured by Infors
Sourced in Switzerland
The Multitron incubator is a laboratory equipment designed for controlled incubation. It provides a stable environment for the cultivation of cell cultures, microorganisms, and other biological samples. The Multitron incubator offers temperature, humidity, and gas control to support the optimal growth conditions required for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Centricon plus 70 centrifugal filter unit, by Merck Group (3 mentions)
Freestyle medium, by Thermo Fisher Scientific (2 mentions)
Qev original sec column sp5, by Izon Science (2 mentions)
Kta avant 25 system, by Cytiva (2 mentions)
Tissue culture flask, by Corning (1 mentions)
Fluostar optima microplate reader, by BMG Labtech (1 mentions)
Li 250a, by LI COR (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Sf 900 2 serum free medium, by Thermo Fisher Scientific (1 mentions)
Expisf cd medium, by Thermo Fisher Scientific (1 mentions)
Vi cell xr, by Beckman Coulter (1 mentions)
Sf9 cell line, by Thermo Fisher Scientific (1 mentions)
Li 180 spectrometer, by LI COR (1 mentions)
Heracell co2 incubator, by Thermo Fisher Scientific (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Via1 cassette, by ChemoMetec (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (1 mentions)
10 protocols using multitron incubator
1
Photoautotrophic Cultivation of Synechococcus
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Synechococcus strains were grown in A+ medium (37 ) containing sodium nitrate (1 g/l), supplemented with zeocin at 100 µg/ml where appropriate. Plates of solid A+ media were made following the addition of 1% agarose and 1 mM sodium thiosulphate. Cultures were grown photoautotrophically in 40 ml of liquid medium in 250 ml baffled flasks under continuous white LED illumination in an Algaetron growth chamber (PSI Instruments) at 200 µmol photons m−2 s−1 at 37°C with shaking at 200 rpm. Transformation plates were incubated at lower irradiances in a Multitron incubator (Infors) with continuous cool white fluorescent illumination at 50–70 µmol photons m−2 s−1 at 30°C. Light irradiance was measured using a Li-Cor Li-250A light sensor equipped with a Li-190SA quantum sensor. To generate growth profiles for the various strains, cells were cultured in 25 cm2 vented Corning tissue-culture flasks and the cell density was determined every 24 h for 7 days by measuring the OD740nm using a Fluostar Optima microplate reader (BMG Labtech).
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Synechococcus strains were grown in A+ medium (37 ) containing sodium nitrate (1 g/l), supplemented with zeocin at 100 µg/ml where appropriate. Plates of solid A+ media were made following the addition of 1% agarose and 1 mM sodium thiosulphate. Cultures were grown photoautotrophically in 40 ml of liquid medium in 250 ml baffled flasks under continuous white LED illumination in an Algaetron growth chamber (PSI Instruments) at 200 µmol photons m−2 s−1 at 37°C with shaking at 200 rpm. Transformation plates were incubated at lower irradiances in a Multitron incubator (Infors) with continuous cool white fluorescent illumination at 50–70 µmol photons m−2 s−1 at 30°C. Light irradiance was measured using a Li-Cor Li-250A light sensor equipped with a Li-190SA quantum sensor. To generate growth profiles for the various strains, cells were cultured in 25 cm2 vented Corning tissue-culture flasks and the cell density was determined every 24 h for 7 days by measuring the OD740nm using a Fluostar Optima microplate reader (BMG Labtech).
2
Exosome Isolation from Cell Culture
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STX-S and STX-N cells were cultured in FreeStyle medium (catalog number 12338018; Thermo Fisher) in a Multitron incubator (Infors HT). Subsequently, cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using either Capricor’s laboratory-scale or large-scale purification method. For the laboratory-scale method, the supernatant was subjected to concentrating filtration using a Centricon Plus-70 centrifugal filter unit (catalog number UFC710008; Millipore) and then subjected to size exclusion chromatography (SEC) using a qEV original SEC column (SP5; Izon). For the large-scale method, the supernatant was subjected to concentrating tangential flow filtration (TFF) on an Äkta Flux s instrument (Cytiva, USA) and then subjected to size exclusion chromatography on an Äkta Avant 25 system (Cytiva, USA).
3
Liquid Cultivation of Spongipellis borealis
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Spongipellis borealis (381) (KC293970) was deposited in the fungal collection at the Department of Biochemistry of Maria Curie-Skłodowska University (Lublin, Poland). The examined microorganism was stored on malt agar slants at 4 °C. The maternal cultures were cultivated for 14 days in a shaking system at 25 °C on liquid Oddoux medium composed of glucose 7 g/L, L-asparagine 0.5 g/L, MgSO4·7H2O, 0.5 g/L, KH2PO4 0.5 g/L, malt extract 8 g/L, casein hydrolysate 1 g/L, thiamine 0.5 mg/L, riboflavin 0.5 mg/L, nicotinamide 0.5 mg/L, pyridoxine hydrochloride 0.5 mg/L, and calcium pantothenate 0.5 mg/L. The maternal mycelium homogenised with a blade homogeniser was used to inoculate the experimental cultures cultivated in 6 L flasks with 3.5 L of media. Then, 13-day submerged liquid fermentation was carried out at 26 °C using a Multitron incubator (Infors, Bottmingen, Switzerland) (agitation rate 120 rpm).
4
Culturing Sf9, ExpiSf9, HEK293T, and Ad293 Cells
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The Sf9 cell line (Thermo Fisher Scientific, Waltham, MA, USA) was cultured in Sf-900 II serum-free medium (SFM; Thermo Fisher Scientific). The ExpiSf9 cell line (Thermo Fisher Scientific) was cultured in ExpiSf CD medium (Thermo Fisher Scientific). Cells were maintained at 28°C, 125 rpm in a Multitron incubator (Infors HT, Annapolis Junction, MD, USA). Both HEK293T (ATCC, Manassas, VA, USA) and Ad293 cell lines (ATCC) were cultured in DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Cell counts and viabilities were measured with a Vi-CELL XR (Beckman Coulter, Brea, CA, USA).
5
Light-Dependent Cell Culture Conditions
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Cells exposed to light were cultivated in a Multitron incubator with CO2 supply (Infors HT, Switzerland). For white light, the built-in LED lights were used for a homogenous illumination from above, resulting in a light intensity of 80 µmol m−2 s−1 (2 mW/cm2) at the height of the cell layer. This intensity was chosen to be as low as possible to minimize the impact on mammalian cells while still allow photosynthetic oxygen production. Red and blue light were provided by an LED strip (LLV-shop, Germany), also providing homogenous illumination from above in the same intensity (measured in µmol m−1 s−1). Samples cultivated in darkness were kept in a HERAcell CO2 incubator (Thermo Fisher Scientific). Standard cell culture conditions (37 °C, 5% CO2) were applied in both incubators. Light spectra were measured using an LI-180 spectrometer (LI-COR Biosciences GmbH, Germany) and are shown in Fig. 1 a.
6
Transient Transfection of Plasmid Constructs in Expi293F Cells
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Plasmid constructs were transiently transfected into Expi293F cells (A14528), using Expi293 Expression Medium (A14351-01), ExpiFectamine 293 Transfection Kit (A14526) and Opti-MEM I Reduced Serum Medium (31985-047) following the Expi293 Expression System transfection protocol (all from ThermoFisher Scientific, Carlsbad, CA, USA). Expi293F cells are human embryonic kidney (HEK) cells derived from the originally described HEK-293 cell line [57 (link)] that has been adapted to suspension growth (293F). Cell number was determined using a NucleoCounter NC-200 automated cell counter (ChemoMetec, Allerod, Denmark; 900-0200) and the Via1-Cassette (ChemoMetec, Allerod, Denmark; 941-0012).
For transient transfections, 1 µg total plasmid DNA was used per ml cell suspension. Accordingly, transfection experiments with 100% RBDmfc-expressing plasmid used 1 µg RBDmfc plasmid DNA (per ml cell suspension), while experiments with 95% and 70% RBDmfc-expressing plasmid used 0.95 and 0.7 µg RBDmfc plasmid DNA (per ml cell suspension), complemented with 0.05 (5%) and 0.3 (30%) µg, respectively, of either empty or PDIA2-expressing plasmid. Transfected cells were incubated for four days in a Multitron incubator (INFORS HT, Bottmingen, Switzerland) at 37 °C, 8% CO2, under shaking at 125 rpm with a 19 mm orbital.
For transient transfections, 1 µg total plasmid DNA was used per ml cell suspension. Accordingly, transfection experiments with 100% RBDmfc-expressing plasmid used 1 µg RBDmfc plasmid DNA (per ml cell suspension), while experiments with 95% and 70% RBDmfc-expressing plasmid used 0.95 and 0.7 µg RBDmfc plasmid DNA (per ml cell suspension), complemented with 0.05 (5%) and 0.3 (30%) µg, respectively, of either empty or PDIA2-expressing plasmid. Transfected cells were incubated for four days in a Multitron incubator (INFORS HT, Bottmingen, Switzerland) at 37 °C, 8% CO2, under shaking at 125 rpm with a 19 mm orbital.
7
Generation of SARS-CoV-2 Spike Expressing Cells
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Human embryonic kidney 293 T cells (293T) were purchased from ATCC (CRL-3216). 293T cells were maintained in culture using Dulbecco’s Modified Eagle Medium (DMEM), high glucose, Glutamax™ containing 10% fetal bovine serum. 293T cells were incubated at 37°C /5% CO2. FreeStyle™ 293F cells (Gibco, 51–0029) were purchased from ThermoFisher. 293F cells were used as a parental cell line to generate spike SARS-CoV-2 delta spike expressing stable cell lines: Stealth X-Spike cells (STX-S). 293F and STX-S cells were maintained in a Multitron incubator (Infors HT) at 37°C, 80% humidified atmosphere with 8% CO2 on an orbital shaker platform rotating at 110 rpm.
8
Exosome Isolation from STX-S Cells
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STX-S cells were cultured in FreeStyle media (ThermoFisher, 12338018) in a Multitron incubator (Infors HT) at 37°C, 80% humidified atmosphere with 8% CO2 on an orbital shaker platform. Subsequently, cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using either Capricor’s lab scale or large-scale purification method. For lab scale: supernatant was subjected to concentrating filtration against a Centricon Plus-70 Centrifugal filter unit (Millipore, UFC710008), then subjected to size exclusion chromatography (SEC) using a qEV original SEC column (Izon, SP5). For large scale: supernatant was subjected to concentrating tangential flow filtration (TFF) on an AKTA Flux s instrument (Cytiva, United States) and then subjected to chromatography on an AKTA Avant 25 (Cytiva, United States).
9
Exosome Isolation from Cell Culture
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STX-S and STX-N cells were cultured in FreeStyle medium (catalog number 12338018; Thermo Fisher) in a Multitron incubator (Infors HT). Subsequently, cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using either Capricor’s laboratory-scale or large-scale purification method. For the laboratory-scale method, the supernatant was subjected to concentrating filtration using a Centricon Plus-70 centrifugal filter unit (catalog number UFC710008; Millipore) and then subjected to size exclusion chromatography (SEC) using a qEV original SEC column (SP5; Izon). For the large-scale method, the supernatant was subjected to concentrating tangential flow filtration (TFF) on an Äkta Flux s instrument (Cytiva, USA) and then subjected to size exclusion chromatography on an Äkta Avant 25 system (Cytiva, USA).
10
Quantification of Volatile Sulfur and Selenium Compounds in Wastewater and Surface Water
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The final DI-SPME-GC/MS method was used to quantify volatile S and Se compounds in raw wastewater from the aerobic and anaerobic sludge reactors of a communal wastewater treatment facility in Dübendorf, Switzerland, and in natural surface water from an ombrotrophic peat bog (Gola di Lago, Switzerland) [50] . Headspace-free samples were collected in triplicate in 10 mL amber glass vials that were immediately sealed with silicone-PTFE septa. A first aliquot of these samples (referred to as 'original aliquots') was analyzed within 12 h of collection (samples stored at 4°C in the dark) to quantify the natural concentrations of volatile S and Se compounds. A second aliquot (referred to as 'incubated aliquots') was spiked with 100 µL of a 100 mM sodium selenite solution to yield a final Se concentrations of ~1mM, placed in a Multitron incubator (Infors-HT, Bottmingen, Switzerland) in the dark at 25°C, and analyzed after 48 h. Before analysis, the DI-SPME-GC/MS method was calibrated (on a daily basis) using aqueous standards with known amounts of analytes and internal standards that were added as described above. The accuracy of the method was verified by spiking measured samples with known amounts of analytes, according to the method of standard addition.
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